Sensors and assays for ubiquitin or ubiquitin-like proteins

ABSTRACT

The present invention provides compositions comprising chimeric polypeptides that bind to free ubiquitin proteins or free ubiquitin-like proteins with high affinity, as well as chimeric polypeptides that bind to both free and conjugated ubiquitin proteins or free and conjugated ubiquitin-like proteins, and methods of using the chimeric polypeptides to determine the amount of free or total ubiquitin or free or total ubiquitin-like proteins in various types of samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/251,871, filed Jan. 18, 2019, now allowed, which is a continuation ofU.S. application Ser. No. 15/977,727, filed May 11, 2018, now issued asU.S. Pat. No. 10,234,459, which is a divisional of U.S. application Ser.No. 14/266,502, filed Apr. 30, 2014, now issued as U.S. Pat. No.10,018,634, which application claims benefit under 35 U.S.C. § 119(e) ofU.S. provisional patent application No. 61/817,517 filed on Apr. 30,2013, the contents of which are incorporated herein by reference intheir entirety.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. GM097452awarded by the National Institutes of Health. The government has certainrights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is CSUV_007_04US_ST25.txt. The text file is 29 KB,created on Apr. 22, 2021, and is being submitted electronically viaEFS-Web.

BACKGROUND OF THE INVENTION

Ubiquitin is a small, highly conserved protein that is found in alleukaryotic cells. Ubiquitin proteins and ubiquitin-like proteinsregulate diverse and vital processes within cells. Ubiquitin proteinshave roles in many cell functions including the mediation of variousstress responses, repair of damaged DNA, regulation of differential geneexpression, and cell cycle control. One of the best characterized rolesof ubiquitin proteins is the regulation of selective protein degradationby the proteasome.

Ubiquitin proteins and ubiquitin-like proteins function through theircovalent attachment to other proteins, which is also referred to in theart as conjugation. The conjugation of a ubiquitin protein or aubiquitin-like protein can influence the target protein in a number ofways which include the signaling of the target protein for degradationby the proteasome, changing the activity of the target protein, orchanging the cellular localization of the target protein. When aubiquitin protein is conjugated to a target protein, the target proteinis said to be ubiquitinated.

Since ubiquitin proteins and ubiquitin-like proteins act through theirconjugation to other proteins, the pools of unconjugated ubiquitin mustbe regulated for the proper functions of virtually allubiquitin-dependent signaling pathways. Despite the importance ofubiquitin and ubiquitin like proteins, few methods to measure freeubiquitin proteins have been described. Typically, these methods havelow precision or are difficult to implement for many labs. What isneeded in the art is a simple, practical assay for free ubiquitinproteins or ubiquitin-like proteins to be measured.

The present invention addresses such needs, providing novel chimericpolypeptide sensors for detecting free ubiquitin proteins orubiquitin-like proteins or, alternatively, both free and conjugatedubiquitin proteins or ubiquitin-like proteins, and related methods touse the chimeric polypeptide sensors to determine the amount of freeubiquitin in a sample and/or the total amount of ubiquitin in a sample.This invention also provides sensitive assays for free ubiquitinproteins or ubiquitin-like proteins, or total ubiquitin proteins orubiquitin-like proteins, in a sample, and can be incorporated inexperiments to measure free or total ubiquitin or ubiquitin-likeproteins in tissues or extracts, and can be used to monitorubiquitination or deubiquitinase enzyme activities in real-time assays.Alternatively, variants of the invention this invention can beincorporated into screens to identify agents that modify ubiquitinconjugation.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions comprising chimericpolypeptides that bind to ubiquitin proteins or ubiquitin-like proteinswith high affinity, and methods of using the chimeric polypeptides todetermine the amount of ubiquitin proteins or ubiquitin-like proteins invarious types of samples. In various embodiments, the chimericpolypeptides bind to only free ubiquitin proteins or free ubiquitin-likeproteins, or bind to both free and conjugated ubiquitin proteins or freeand conjugated ubiquitin-like proteins.

In some embodiments, the invention provides a chimeric polypeptidecomprising: two or more polypeptide sequences that bind a ubiquitinprotein or a ubiquitin-like protein, wherein the sequences bindnon-overlapping regions of the same ubiquitin protein or ubiquitin-likeprotein, and one or more linker(s), wherein each linker connects two ormore of the sequences. In some embodiments, the chimeric polypeptidepreferentially binds to the free ubiquitin protein or the freeubiquitin-like protein as compared to the conjugated ubiquitin proteinor the conjugated ubiquitin-like protein. In some embodiments, thechimeric polypeptide binds free and conjugated ubiquitin.

In some embodiments, the invention comprises a chimeric polypeptide,wherein the chimeric polypeptide comprises three polypeptide sequencesthat bind to non-overlapping regions of the ubiquitin protein or theubiquitin-like protein, wherein the three polypeptide sequences areconnected by two linkers.

In some embodiments, the invention provides a chimeric polypeptide thatcomprises, in the following order (amino terminal to carboxy terminal):a first polypeptide sequence that binds to a first region of theubiquitin protein or the ubiquitin-like protein, a first linker thatconnects the first polypeptide sequence with a second polypeptidesequence, the second polypeptide sequence, which binds to a secondregion of the ubiquitin protein or the ubiquitin-like protein, a secondlinker that connects the second polypeptide sequence with a thirdpolypeptide sequence; and the third polypeptide sequence, which binds toa third region of the ubiquitin protein or the ubiquitin likepolypeptide, wherein the regions of the ubiquitin protein or theubiquitin-like protein bound by the first, second, and third polypeptidesequences are non-overlapping. In some embodiments, the chimericpolypeptide comprises linker(s), wherein the linkers are polypeptides.

In some embodiments, the invention provides the chimeric polypeptide,wherein the sequences comprise a sequence that binds to the ubiquitin Cterminus. In some embodiments, the chimeric polypeptide comprises asequence that binds to the ubiquitin hydrophobic patch. In someembodiments, the chimeric polypeptide comprises a sequence that binds tothe surface around Asp58 of ubiquitin. In some embodiments, the chimericpolypeptide comprises a sequence that binds to the ubiquitin C terminusand a sequence that binds to the ubiquitin hydrophobic patch. In someembodiments the chimeric polypeptide comprises a sequence that binds tothe ubiquitin C terminus and a sequence that binds the surface aroundAsp58 of ubiquitin. In some embodiments, the chimeric polypeptidecomprises a sequence that binds to the ubiquitin hydrophobic patch and asequence that binds to surface around Asp58 of ubiquitin. In someembodiments, the chimeric polypeptide comprises a sequence that binds tothe ubiquitin C terminus, a sequence that binds to the ubiquitinhydrophobic patch, and a sequence that binds to the surface around Asp58of ubiquitin.

In some embodiments, the invention provides a chimeric polypeptide,wherein the sequences comprise the zinc finger binding domain ofIsopeptidase T. In some embodiments, the chimeric polypeptide comprisessequences comprising a Ruz domain of Rabex-5. In some embodiments, thechimeric polypeptide comprises sequences comprising a ubiquitininteracting motif of Vps27. In some embodiments, the chimericpolypeptide comprises sequences comprising the ubiquitin associateddomain of Dsk2. In some embodiments, the chimeric polypeptide comprisessequences comprising a zinc finger binding domain of Isopeptidase T andthe Ruz domain of Rabex-5. In some embodiments, the chimeric polypeptidecomprises sequences comprising the zinc finger binding domain ofIsopeptidase T and the ubiquitin interacting motif of Vps27. In someembodiments, the chimeric polypeptide comprises sequences comprising aRuz domain of Rabex-5 and the ubiquitin interacting motif of Vps27. Insome embodiments, the invention provides a chimeric polypeptide, whereinthe sequences comprise the zinc finger binding domain of Isopeptidase T,the Ruz domain of Rabex-5, and the ubiquitin interacting motif of Vps27.

In some embodiments, the chimeric polypeptide comprises sequencescomprising the zinc finger binding domain of Isopeptidase T and theubiquitin associated domain of Dsk2. In some embodiments, the chimericpolypeptide comprises sequences comprising the Ruz domain of Rabex-5 andthe ubiquitin associated domain of Dsk2. In some embodiments, thechimeric polypeptide comprises sequences comprising the zinc fingerbinding domain of Isopeptidase T, the Ruz domain of Rabex-5, and theubiquitin associated domain of Dsk2.

In some embodiments, the invention provides the chimeric polypeptide,wherein the sequences bind to non-overlapping regions of the ubiquitinprotein. In some embodiments, the chimeric polypeptide comprisessequences bind to non-overlapping regions of the ubiquitin-like protein.In some embodiments, the ubiquitin-like protein is Nedd8. In someembodiments, the ubiquitin-like protein is SUMO.

In some embodiments, the invention provides the chimeric polypeptidecomprising a detectable label attached to the polypeptide. In someembodiments, the detectable label is a fluorophore. In some embodiments,the chimeric polypeptide comprises a quencher that is attached to thechimeric polypeptide.

In some embodiments, the invention provides a method of determining anamount of a free ubiquitin protein or a free ubiquitin-like protein in asample, the method comprising: contacting the sample with a chimericpolypeptide that preferentially binds to the free ubiquitin protein orthe free ubiquitin-like protein as compared the conjugated ubiquitinprotein or the conjugated ubiquitin-like protein for a period of time,wherein the chimeric polypeptide comprises two or more polypeptidesequences that bind the ubiquitin protein or the ubiquitin-like protein,wherein the sequences bind non-overlapping regions of the ubiquitinprotein or ubiquitin-like protein; and one or more linker(s), whereineach linker(s) connect two or more of the sequences; and determining:(i) an amount of the free ubiquitin protein or free ubiquitin-likeprotein bound to the chimeric polypeptide; or (ii) an amount of a freecompetitor protein bound to the chimeric polypeptide, wherein if step(ii) is performed, the method further comprises step contacting thesample with the free competitor ubiquitin protein or the free competitorubiquitin-like protein prior to step determining an amount of freecompetitor protein bound to the chimeric polypeptide, therebydetermining the amount of the free ubiquitin protein or the freeubiquitin-like protein in the sample.

In some embodiments, the method comprises contacting the sample with thechimeric polypeptide for the period of time, and determining the amountof the free ubiquitin protein or free ubiquitin-like protein bound tothe chimeric polypeptide.

In some embodiments, the chimeric polypeptide is the chimericpolypeptide is any chimeric peptide disclosed by the invention. In someembodiments, the method the chimeric polypeptide is tagged with adetectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound free ubiquitin protein or the bound free ubiquitin-likeprotein to a predetermined value or to a control value.

In some embodiments, the determination of an amount of the freeubiquitin protein or free ubiquitin-like protein bound to the chimericpolypeptide further comprises determining the amount of the freeubiquitin competitor protein or the free ubiquitin-like competitorprotein bound to the chimeric polypeptide at two or more time pointsduring the period of time. In some embodiments, determining an amount ofthe free ubiquitin protein or free ubiquitin-like protein bound to thechimeric polypeptide comprises determining the amount of the freeubiquitin competitor protein or the free ubiquitin-like competitorprotein bound to the chimeric polypeptide at two or more regular timepoints throughout the period of time. In some embodiments, the methodfurther comprises comparing the amount of the bound free ubiquitinprotein or the bound free ubiquitin-like protein to a predeterminedvalue or to a control value at two or more time points during the periodof time.

In some embodiments, the sample is a biological sample. In someembodiments, the biological sample is derived from cultured cells,optionally eukaryotic cells. In some embodiments, the biological sampleis derived from a biological tissue, and wherein the biological tissueoptionally comprises eukaryotic cells.

In some embodiments, the method of determining an amount of a freeubiquitin protein or a free ubiquitin-like protein in a sample comprisescontacting the sample with the chimeric polypeptide that preferentiallybinds to the free ubiquitin protein or the free ubiquitin-like proteinas compared the conjugated ubiquitin protein or the conjugatedubiquitin-like protein for the period of time, contacting the samplewith the free competitor ubiquitin protein or the free competitorubiquitin-like protein, and determining the amount of the freecompetitor ubiquitin protein or the free competitor ubiquitin-likeprotein bound to the chimeric polypeptide.

In some embodiments, the chimeric polypeptide is any chimeric peptidedisclosed by the invention. In some embodiments, the method the chimericpolypeptide is tagged with a detectable label. In some embodiments, thedetectable label is a fluorophore. In some embodiments, the methodcomprises detecting the detectable label. In some embodiments, thedetection comprises measuring fluorescence intensity. In someembodiments, the detection comprises measuring fluorescence anisotropy.

In some embodiments, the further comprises comparing the amount of thefree competitor ubiquitin protein or free competitor ubiquitin-likeprotein bound to the chimeric polypeptide to a predetermined value or acontrol value.

In some embodiments, determining the amount of the free competitorubiquitin protein or the free competitor ubiquitin-like protein bound tothe chimeric polypeptide comprises determining the amount of theubiquitin competitor protein or the ubiquitin-like competitor proteinbound to the chimeric polypeptide at two or more time points during theperiod of time. In some embodiments, determining the amount of the freecompetitor ubiquitin protein or the free competitor ubiquitin-likeprotein bound to the chimeric polypeptide comprises determining theamount of the ubiquitin competitor protein or the ubiquitin-likecompetitor protein bound to the chimeric polypeptide at two or moreregular time points throughout the period of time.

In some embodiments, the method further comprises comparing the amountof the ubiquitin competitor protein or the ubiquitin-like competitorprotein bound to the chimeric polypeptide to a predetermined value or acontrol value at two or more time points during the period of time.

In some embodiments, the free ubiquitin competitor protein or the freeubiquitin-like competitor protein is tagged with a first detectablelabel, and the chimeric polypeptide is tagged with a second detectablelabel.

In some embodiments, the first detectable label and the seconddetectable label are suitable for performing fluorescence resonanceenergy transfer (FRET). In some embodiments the first detectable labeland the second detectable label are the same. In some embodiments, thefirst detectable label and the second detectable label are different. Insome embodiments, the first detectable label is a fluorophore. In someembodiments, the second detectable label is a fluorophore. In someembodiments, the first detectable label is a quencher and the seconddetectable label is a fluorophore. In some embodiments, the firstdetectable label is a fluorophore and the second detectable label is aquencher. In some embodiments, determining the amount of the freecompetitor ubiquitin protein or the free competitor ubiquitin-likeprotein bound to the chimeric polypeptide comprises detecting thedetectable labels. In some embodiments, the detection comprisesmeasuring fluorescence intensity. In some embodiments, the detectioncomprises measuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound ubiquitin protein or bound ubiquitin-like protein to apredetermined value or a control value. In some embodiments, determiningthe amount of the free competitor ubiquitin protein or the freecompetitor ubiquitin-like protein bound to the chimeric polypeptidecomprises determining the amount of the free ubiquitin competitorprotein or the free ubiquitin-like competitor protein bound to thechimeric polypeptide at two or more time points during the period oftime. In some embodiments, determining the amount of the free competitorubiquitin protein or the free competitor ubiquitin-like protein bound tothe chimeric polypeptide comprises determining the amount of theubiquitin competitor protein or the ubiquitin-like competitor proteinbound to the chimeric polypeptide at two or more regular time pointsthroughout the period of time In some embodiments, the method furthercomprises comparing the amount of the ubiquitin competitor protein orthe ubiquitin-like competitor protein bound to the chimeric polypeptideto a predetermined value or a control value at two or more time pointsduring the period of time.

In some embodiments, the invention provides a method of determining atotal amount of a ubiquitin protein or a conjugated ubiquitin-likeprotein in a sample, the method comprising contacting the sample with achimeric polypeptide that binds to both the free and conjugatedubiquitin protein or both the free and conjugated ubiquitin-like proteinfor a period of time, wherein the chimeric polypeptide comprises two ormore polypeptide sequences that bind the ubiquitin protein or theubiquitin-like protein, wherein the sequences bind non-overlappingregions of the ubiquitin protein or ubiquitin-like protein, and one ormore linker(s), wherein each linker(s) connects two or more of thesequences; and determining: an amount of the ubiquitin protein orubiquitin-like protein bound to the chimeric polypeptide; or an amountof a competitor ubiquitin protein or a competitor ubiquitin-like proteinbound to the chimeric polypeptide, wherein if an amount of a competitorubiquitin protein or a competitor ubiquitin-like protein bound to thechimeric polypeptide is determined, the method further comprises stepcontacting the chimeric polypeptide sensor or the sample with thecompetitor ubiquitin protein or the competitor ubiquitin-like proteinprior to step determining an amount of a competitor ubiquitin protein ora competitor ubiquitin-like protein bound to the chimeric polypeptide,thereby determining the total amount of the conjugated ubiquitin proteinor the conjugated ubiquitin-like protein in the sample.

In some embodiments, the method comprises contacting the sample with thechimeric polypeptide for the period of time, and determining the amountof the ubiquitin protein or ubiquitin-like protein bound to the chimericpolypeptide.

In some embodiments, the chimeric polypeptide is the chimericpolypeptide is any chimeric peptide disclosed by the invention. In someembodiments, the method the chimeric polypeptide is tagged with adetectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound ubiquitin protein or the bound ubiquitin-like protein to apredetermined value or to a control value. In some embodiments, themethod comprises determining the amount of the bound ubiquitin proteinor the bound ubiquitin-like protein at two or more regular time pointsthroughout the period of time. In some embodiments, the method comprisescomparing the amount of the bound ubiquitin protein or the boundubiquitin-like protein to a predetermined value or to a control value attwo or more time points during the period of time.

In some embodiments, the sample is a biological sample. In someembodiments, the biological sample is derived from cultured cells,optionally eukaryotic cells. In some embodiments, the biological sampleis derived from a biological tissue.

In some embodiments, the method comprises contacting the sample with thechimeric polypeptide for the period of time, determining an amount ofthe competitor ubiquitin protein or the conjugated competitorubiquitin-like protein bound to the chimeric polypeptide, and contactingthe sample or the chimeric polypeptide sensor with the competitorubiquitin protein or the conjugated competitor ubiquitin-like proteinprior to determining an amount of the competitor ubiquitin protein orthe conjugated competitor ubiquitin-like protein bound to the chimericpolypeptide.

In some embodiments, the chimeric polypeptide is any chimeric peptidedisclosed by the invention. In some embodiments, the method the chimericpolypeptide is tagged with a detectable label. In some embodiments, thedetectable label is a fluorophore. In some embodiments, the methodcomprises detecting the detectable label. In some embodiments, thedetection comprises measuring fluorescence intensity. In someembodiments, the detection comprises measuring fluorescence anisotropy.

In some embodiments, the method further comprises: comparing the amountof the competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide to a predetermined value or acontrol value.

In some embodiments, determining an amount of the competitor ubiquitinprotein or the conjugated competitor ubiquitin-like protein bound to thechimeric polypeptide comprises determining the amount of the competitorubiquitin protein or the competitor ubiquitin-like protein bound to thechimeric polypeptide at two or more time points during the period oftime. In some embodiments, determining an amount of the competitorubiquitin protein or the conjugated competitor ubiquitin-like proteinbound to the chimeric polypeptide comprises determining the amount ofthe competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide at two or more regular timepoints throughout the period of time.

In some embodiments, the method further comprises comparing the amountof the competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide to a predetermined value or acontrol value at two or more time points during the period of time.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a first detectable label, and thechimeric polypeptide is tagged with a second detectable label. In someembodiments, the first detectable label and the second detectable labelare suitable for performing fluorescence resonance energy transfer(FRET). In some embodiments, the first detectable label and the seconddetectable label are the same. In some embodiments, the first detectablelabel and the second detectable label are different. In someembodiments, the first detectable label is a fluorophore. In someembodiments, the second detectable label is a fluorophore. In someembodiments, the first detectable label is a quencher and the seconddetectable label is a fluorophore. In some embodiments, the firstdetectable label is a fluorophore and the second detectable label is aquencher. In some embodiments, the method comprises detecting thedetectable labels. In some embodiments, the detection comprisesmeasuring fluorescence intensity. In some embodiments, the detectioncomprises measuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound ubiquitin protein or bound ubiquitin-like protein to apredetermined value or a control value. In some embodiments, the methodcomprises determining the amount of the competitor ubiquitin protein orthe competitor ubiquitin-like protein bound to the chimeric polypeptideat two or more time points during the period of time.

In some embodiments, the method comprises determining the amount of thecompetitor ubiquitin protein or the competitor ubiquitin-like proteinbound to the chimeric polypeptide at two or more regular time pointsthroughout the period of time. In some embodiments, the method furthercomprises comparing the amount of the competitor ubiquitin protein orthe competitor ubiquitin-like protein bound to the chimeric polypeptideto a predetermined value or a control value at two or more time pointsduring the period of time.

In some embodiments, the invention provides a method of determining adeubiquitinase activity of a known or candidate deubiquitinase,comprising providing a mixture, comprising a chimeric polypeptide,wherein the chimeric polypeptide preferentially binds to a freeubiquitin protein or a free ubiquitin-like protein as compared to theconjugated ubiquitin protein or the conjugated ubiquitin-like proteincomprising two or more polypeptide sequences that bind a ubiquitinprotein or a ubiquitin-like protein, wherein the sequences bindnon-overlapping regions of the ubiquitin protein or ubiquitin-likeprotein, and one or more linkers, wherein each linker connects two ormore of the sequences; and a conjugated ubiquitin; contacting themixture with a known or candidate deubiquitinase for a period of time;and determining an amount of free ubiquitin in the mixture, therebydetermining the deubiquitinase activity of the known or candidatedeubiquitinase.

In some embodiments, the method further comprises, comparing the amountof the free ubiquitin determined to the amount of the free ubiquitindetermined prior to contacting the mixture with a known or candidatedeubiquitinase for a period of time or determining the amount of freeubiquitin in a negative control, wherein a greater amount of freeubiquitin after contact with the known or candidate deubiquitinaseindicates that the known or candidate deubiquitinase decreasesconjugation of the ubiquitin protein or the ubiquitin-like protein, anda lesser amount of free ubiquitin associated with the presence of theknown or candidate deubiquitinase indicates that the known or candidatedeubiquitinase increases the conjugation of the ubiquitin protein or theubiquitin-like protein.

In some embodiments, the method further comprises comparing the amountof free ubiquitin determined at a first timepoint to the amount of freeubiquitin determined at one or more subsequent time point(s), wherein agreater amount of free ubiquitin determined at the one or moresubsequent time point(s) indicates that the known deubiquitinase or thecandidate deubiquitinase is a deubiquitinase of the ubiquitin protein orthe ubiquitin-like protein.

In some embodiments, the method comprises, providing a plurality ofindependently addressable mixtures, each comprising the chimericpolypeptide and a different conjugated ubiquitin; contacting each of themixtures with the known deubiquitinase or the candidate deubiquitinasefor a period of time, and determining the amount of the free ubiquitinprotein or free ubiquitin-like protein bound to the chimeric polypeptidein each of the mixtures, thereby determining the deubiquinase activityof the known deubiquitinase or candidate deubiquitinase for each of thedifferent ubiquitins in the mixtures.

In some embodiments, the chimeric polypeptide is the chimericpolypeptide is any chimeric peptide disclosed by the invention. In someembodiments, the method the chimeric polypeptide is tagged with adetectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound free ubiquitin protein or the bound free ubiquitin-likeprotein to a predetermined value or to a control value. In someembodiments, the method further comprises comparing the amount of thebound free ubiquitin protein or the bound free ubiquitin-like protein toa predetermined value or to a control value at two or more time pointsduring the period of time.

In some embodiments, the chimeric polypeptide is the chimericpolypeptide is any chimeric peptide disclosed by the invention. In someembodiments, the method the chimeric polypeptide is tagged with adetectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide to a predetermined value or acontrol value. In some embodiments, the method further comprisescomparing the amount of the competitor ubiquitin protein or thecompetitor ubiquitin-like protein bound to the chimeric polypeptide to apredetermined value or a control value at two or more time points duringthe period of time.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a first detectable label, and thechimeric polypeptide is tagged with a second detectable label. In someembodiments, the first detectable label and the second detectable labelare suitable for performing fluorescence resonance energy transfer(FRET). In some embodiments, the first detectable label and the seconddetectable label are the same. In some embodiments, the first detectablelabel and the second detectable label are different. In someembodiments, the first detectable label is a fluorophore. In someembodiments, the second detectable label is a fluorophore. In someembodiments, the first detectable label is a quencher and the seconddetectable label is a fluorophore. In some embodiments, the firstdetectable label is a fluorophore and the second detectable label is aquencher. In some embodiments, the method comprises detecting thedetectable labels. In some embodiments, the detection comprisesmeasuring fluorescence intensity. In some embodiments, the detectioncomprises measuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide to a predetermined value or acontrol value at two or more time points during the period of time.

In some embodiments, the invention provides a method of identifying anagent that modulates the conjugation of a ubiquitin protein or aubiquitin-like protein to a substrate, the method comprising contactinga mixture comprising a ubiquitin substrate or a ubiquitin-like proteinsubstrate with the ubiquitin protein or the ubiquitin-like protein and acandidate agent for a period of time; contacting the ubiquitin proteinor the ubiquitin-like protein with a chimeric polypeptide, wherein thechimeric polypeptide preferentially binds to the free ubiquitin proteinor the free ubiquitin-like protein as compared to the conjugatedubiquitin protein or the conjugated ubiquitin-like protein comprising:two or more polypeptide sequences that bind a ubiquitin protein or aubiquitin-like protein, wherein the sequences bind non-overlappingregions of the ubiquitin protein or ubiquitin-like protein and one ormore linkers, wherein each linker connects two or more of the sequences;determining an amount of the free ubiquitin protein or freeubiquitin-like protein bound to the chimeric polypeptide; or an amountof a competitor ubiquitin protein or a competitor ubiquitin-like proteinbound to the chimeric polypeptide, wherein if an amount of a competitorubiquitin protein or a competitor ubiquitin-like protein bound to thechimeric polypeptide is determined, the method further comprisescontacting the chimeric polypeptide sensor with the competitor ubiquitinprotein or the competitor ubiquitin-like protein prior determining theamount of a competitor ubiquitin protein or a competitor ubiquitin-likeprotein bound to the chimeric polypeptide.

In some embodiments, the method comprises contacting the mixturecomprising the ubiquitin substrate or the ubiquitin-like proteinsubstrate with the ubiquitin protein or the ubiquitin-like protein andthe candidate agent, contacting the ubiquitin protein or theubiquitin-like protein with the chimeric polypeptide, determining anamount of the free ubiquitin protein or free ubiquitin-like proteinbound to the chimeric polypeptide; and comparing the amount of the freeubiquitin determined to the amount of the free ubiquitin determined whenusing a negative control instead of the candidate agent, wherein agreater amount of free ubiquitin associated with the presence of thecandidate agent indicates that the candidate agent decreases conjugationof the ubiquitin protein or the ubiquitin-like protein, and wherein alesser amount of free ubiquitin associated with the presence of thecandidate agent indicates that the candidate agent increases theconjugation of the ubiquitin protein or the ubiquitin-like protein.

In some embodiments, the method comprises contacting the mixturecomprising the ubiquitin substrate or the ubiquitin-like proteinsubstrate with the ubiquitin protein or the ubiquitin-like protein andthe candidate agent; contacting the ubiquitin protein or theubiquitin-like protein with the chimeric polypeptide; determining anamount of the competitor ubiquitin protein or the competitorubiquitin-like protein bound to the chimeric polypeptide; and contactingthe chimeric polypeptide sensor with the competitor ubiquitin protein orthe competitor ubiquitin-like protein prior to determining an amount ofthe competitor ubiquitin protein or the competitor ubiquitin-likeprotein bound to the chimeric polypeptide; comparing the amount of thebound competitor protein determined to the amount of the boundcompetitor protein determined when using a negative control instead ofthe candidate agent, wherein a greater amount of the bound competitorprotein associated with the presence of the candidate agent indicatesthat the candidate agent increases conjugation of the ubiquitin proteinor the ubiquitin-like protein, and wherein a lesser amount of the boundcompetitor protein associated with the presence of the candidate agentindicates that the candidate agent decreases the conjugation of theubiquitin protein or the ubiquitin-like protein.

In some embodiments, the chimeric polypeptide is the chimericpolypeptide is any chimeric peptide disclosed by the invention. In someembodiments, the method the chimeric polypeptide is tagged with adetectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method comprises determining the amount of thecompetitor ubiquitin protein or the competitor ubiquitin-like proteinbound to the chimeric polypeptide at two or more time points during theperiod of time. In some embodiments, the method comprises determiningthe amount of the competitor ubiquitin protein or the competitorubiquitin-like protein bound to the chimeric polypeptide at two or moreregular time points throughout the period of time.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a detectable label. In someembodiments, the detectable label is a fluorophore. In some embodiments,the method comprises detecting the detectable label. In someembodiments, the detecting comprises measuring fluorescence intensity.In some embodiments, the detecting comprises measuring fluorescenceanisotropy.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a first detectable label, and thechimeric polypeptide is tagged with a second detectable label. In someembodiments, the first detectable label and the second detectable labelare suitable for performing fluorescence resonance energy transfer(FRET). In some embodiments, the first detectable label and the seconddetectable label are the same. In some embodiments, the first detectablelabel and the second detectable label are different. In someembodiments, the first detectable label is a fluorophore. In someembodiments, the second detectable label is a fluorophore. In someembodiments, the first detectable label is a quencher and the seconddetectable label is a fluorophore. In some embodiments, the firstdetectable label is a fluorophore and the second detectable label is aquencher. In some embodiments, the method comprises detecting thedetectable labels. In some embodiments, the detection comprisesmeasuring fluorescence intensity. In some embodiments, the detectioncomprises measuring fluorescence anisotropy.

In some embodiments, the method comprises determining the amount of thecompetitor ubiquitin protein or the competitor ubiquitin-like proteinbound to the chimeric polypeptide at two or more time points during theperiod of time. In some embodiments, the method comprises determiningthe amount of the competitor ubiquitin protein or the competitorubiquitin-like protein bound to the chimeric polypeptide at two or moreregular time points throughout the period of time.

In some embodiments, the invention provides a method of determining anamount of an intact extracellular ubiquitin protein in a samplecontaining serum, the method comprising: contacting the sample with achimeric polypeptide that preferentially binds to the intactextracellular ubiquitin protein, wherein the chimeric polypeptidecomprises: two or more polypeptide sequences that bind the ubiquitinprotein or the ubiquitin-like protein, wherein the sequences comprise asequence that binds to the ubiquitin C-terminus or the ubiquitin-likeprotein C-terminus and wherein the sequences bind non-overlappingregions of the ubiquitin protein or the ubiquitin-like protein; and oneor more linker(s), wherein the linker(s) connect two or more of thesequences; and determining an amount of the intact extracellularubiquitin protein bound to the chimeric polypeptide; or an amount of acompetitor ubiquitin protein or a competitor ubiquitin-like proteinbound to the chimeric polypeptide, wherein if the amount of a competitorubiquitin protein or a competitor ubiquitin-like protein bound to thechimeric polypeptide is determined, the method further comprisescontacting the sample with the competitor ubiquitin protein or thecompetitor ubiquitin-like protein prior to determining an amount of acompetitor ubiquitin protein or a competitor ubiquitin-like proteinbound to the chimeric polypeptide, thereby determining the amount of theintact extracellular ubiquitin protein in the sample.

In some embodiments, the method comprises contacting the sample with thechimeric polypeptide for the period of time; and determining the amountof the intact extracellular ubiquitin protein bound to the chimericpolypeptide.

In some embodiments, the method the chimeric polypeptide is tagged witha detectable label. In some embodiments, the detectable label is afluorophore. In some embodiments, the method comprises detecting thedetectable label. In some embodiments, the detection comprises measuringfluorescence intensity. In some embodiments, the detection comprisesmeasuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound intact extracellular ubiquitin protein to a predeterminedvalue or to a control value. In some embodiments, the method comprisescontacting the sample with the chimeric polypeptide for the period oftime determining the amount of the competitor protein bound to thechimeric polypeptide.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a detectable label. In someembodiments, the detectable label is a fluorophore. In some embodiments,the method comprises detecting the detectable label. In someembodiments, the detecting comprises measuring fluorescence intensity.In some embodiments, the detecting comprises measuring fluorescenceanisotropy.

In some embodiments, the method further comprises, comparing the amountof the competitor ubiquitin protein or competitor ubiquitin-like proteinbound to the chimeric polypeptide to a predetermined value or a controlvalue.

In some embodiments, the competitor ubiquitin protein or the competitorubiquitin-like protein is tagged with a first detectable label, and thechimeric polypeptide is tagged with a second detectable label. In someembodiments, the first detectable label and the second detectable labelare suitable for performing fluorescence resonance energy transfer(FRET). In some embodiments, the first detectable label and the seconddetectable label are the same. In some embodiments, the first detectablelabel and the second detectable label are different. In someembodiments, the first detectable label is a fluorophore. In someembodiments, the second detectable label is a fluorophore. In someembodiments, the first detectable label is a quencher and the seconddetectable label is a fluorophore. In some embodiments, the firstdetectable label is a fluorophore and the second detectable label is aquencher. In some embodiments, the method comprises detecting thedetectable labels. In some embodiments, the detection comprisesmeasuring fluorescence intensity. In some embodiments, the detectioncomprises measuring fluorescence anisotropy.

In some embodiments, the method further comprises comparing the amountof the bound ubiquitin protein or bound ubiquitin-like protein to apredetermined value or a control value.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

DESCRIPTIONS OF THE FIGURES

FIG. 1 provides a schematic illustration of the design concept for thechimeric polypeptide sensors for free ubiquitin. Ubiquitin is depictedinteracting with three binding domains that bind non-overlapping regionsof ubiquitin (top). An example chimeric polypeptide comprising threedomains connected by two linkers is depicted binding to ubiquitin atthree non-overlapping sites (bottom).

FIG. 2 provides a construction scheme for the chimeric polypeptide.Here, the depicted polypeptide has 3 domains, a domain that binds theubiquitin C terminus, a domain that binds the ubiquitin hydrophobicpatch, and a domain that binds to the surface of ubiquitin near Asp58,that are connected by two linkers.

FIG. 3 provides schematic diagrams of two prototype chimericpolypeptides comprising three binding domains that bind ubiquitin innon-overlapping regions. Both prototype polypeptides contain a domainthat binds the ubiquitin C terminus, a domain that binds the ubiquitinhydrophobic patch, and a domain that binds to the surface of ubiquitinnear Asp58. The binding domains are connected by polypeptide linkers. Inthe first prototype (tIVR) the domain that binds to the C terminus ofubiquitin is the zinc finger domain of Isopeptidase T (IsoT^(ZnF)), thedomain that binds to the ubiquitin hydrophobic patch is theubiquitin-interaction motif from the Vps27 protein (Vps27^(UIM)), andthe domain that binds to the surface of ubiquitin near Asp58 is theRabex-5 ubiquitin binding zinc finger (Ruz). In the second prototype(tIDR), the IsoT^(ZnF) and Ruz domains are linked to the ubiquitinassociated (UBA) domain of the Dsk2 protein (Dsk2^(UBA)), which binds tothe ubiquitin hydrophobic patch. Below the schematics, the amino acidsequences of Vps27^(UIM), Ruz, IsoT^(ZnF), and Dsk2^(UBA) are shown inthe tIVR and tIDR chimeric polypeptides.

FIG. 4 shows structural models of tIVR and tIDR bound to ubiquitin.

FIG. 5 provides the affinities of tIVR and tIDR to fluorophore-taggedubiquitin. The values were calculated from assays incorporatingfluorophore-labeled ubiquitin. Experiments were performed withfluorescein, Atto532, and Alexa488-tagged ubiquitin tagged with theindicated fluorophore at residue 20. The experimentally determined K_(d)values are presented in the table.

FIG. 6 shows the binding curves of tIVR and tIDR to free ubiquitin.Affinity of tIVR to free ubiquitin was measured by detectingfluorescence anisotropy. Different concentrations of free ubiquitin wereadded to 150 pM (top, left). Affinity between Alexa488-tagged ubiquitinand Atto532-tagged was measured by detecting change in fluorescenceintensity. Different concentrations of tIVR were added to 50 pMAtto532-tagged ubiquitin or 100 pM Alexa488-tagged tagged ubiquitin(bottom, left). Binding between fluorescein tagged ubiquitin and tIDRalso was measured as the ratio of association and dissociation rates(right panel). Changes in fluorescence anisotropy of 50 nM fluoresceintagged ubiquitin were measured with and without 26 nM tIDR to measureassociation. Dissociation of 50 nM tIDR from 50 nM fluorescein taggedubiquitin was measured by detecting change in fluorescence anisotropy.

FIG. 7 shows a table and graphs showing that various chimericpolypeptide sensors with different ubiquitin binding domains havedifferent affinities for free ubiquitin. The affinities (as K_(d)values) of Atto532-tagged chimeric polypeptides comprising one, two, orthree ubiquitin binding domains are displayed in the table (top). Thegraphs show the fluorescence intensities of mixtures of theAtto532-tagged chimeric peptides measured as a function of freeubiquitin concentration; the lines are fits to single-site bindingisotherms (bottom). Curves generated from experiments such as these canbe used as a calibration standard in assays to measure free ubiquitinproteins in complex biological samples.

FIG. 8 provides a graph and a table showing the affinity of tIVR to freeubiquitin, conjugated ubiquitin and Nedd8. The tIVR polypeptide has anapproximately 3000-fold higher affinity for free ubiquitin than forconjugated ubiquitin or free Nedd8. A competition assay was performed todetermine the binding affinity of tIVR to free ubiquitin, conjugatedubiquitin, or Nedd8 (top). When bound to tIVR, the Atto532-taggedubiquitin display reduced fluorescent intensity when stimulated comparedto Atto532-tagged ubiquitin that is unbound to tIVR. Fluorescenceintensity at 551 nm was measured in counts per second (CPS). Thecompetitor proteins, free ubiquitin (circles), conjugated ubiquitin(triangles), or Nedd8 (squares) were added to a 10 nM tIVR and 10 nMAtto532-tagged ubiquitin mixture. Since Atto532-tagged ubiquitindisplaced from tIVR has enhanced fluorescence emission, increasedfluorescence intensity indicates binding of competitor proteins to tIVR.A table lists the Ki values for tIVR for free ubiquitin, conjugatedubiquitin, and Nedd8 (bottom).

FIG. 9 provides a graph and a table showing that tIVR has similarbinding affinities for different kinds of conjugated free ubiquitin. Acompetition assay was performed testing 0.8 nM Atto532 tagged ubiquitinand 6 nM tIVR. Fluorescence intensity was measured as differentconcentrations of different linkage types of conjugated ubiquitinproteins were added (top). The binding affinities, shown as Ki values,of each linkage type of di- or poly-ubiquitin are listed in the table(bottom).

FIG. 10 provides a diagram of tIVR with normal length or shortenedlinker and binding with conjugated ubiquitin and a table reporting thebinding properties of tIVR with a shortened linker. Reducing the lengthof the linker connecting the domain that binds the ubiquitin C terminuswith the domain that binds the ubiquitin hydrophobic patch increases thebinding specificity of tIVR to free ubiquitin compared to conjugatedubiquitin.

FIG. 11 provides a table demonstrating that an optimized linker 1 intIVR is short and made more rigid by having a high fraction of Alaresidues. The table lists K_(d) to Atto532-tagged ubiquitin and Ub-GB1 amodel of conjugated ubiquitin, and ratio of affinities for freeubiquitin over conjugated ubiquitin, for a short tIVR linker 1 (SEQ IDNO: 30), a different short linker 1 for tIVR (SEQ ID NO: 13), or a longtIVR linker 1 (SEQ ID: 28).

FIG. 12 provides graphs that evaluate the binding of Atto532-taggedubiquitin and Ub-GB1 to tIVR with linker 1 variants. Differentconcentrations of tIVR with a short linker 1 (SEQ ID NO: 30) are addedto a mixture containing 1 nM atto532 tagged ubiquitin (left). Differentconcentrations of tIVR containing a long linker 1 (SEQ ID NO: 28) areadded to a mixture containing 0.5 nM Atto532-tagged ubiquitin (middle).Different concentrations of Ub-GB1 are added to a mixture containing 3nM Atto532-tagged ubiquitin and 8 nM tIVR with a long linker 1 (SEQ IDNO: 28; right).

FIG. 13 provides illustrations of how the chimeric peptide sensors canbe used with assays that measure fluorescence intensity. When chimericpolypeptide sensors bind a fluorophore-tagged ubiquitin protein, thefluorescence intensity is reduced compared to when thefluorophore-tagged ubiquitin is unbound (left). When afluorophore-tagged chimeric polypeptide sensor is bound to a ubiquitinprotein, the fluorescence intensity is increased compared to when thechimeric polypeptide is unbound (right).

FIG. 14 provides graphs showing that unbound fluorophore-taggedubiquitins have altered fluorescence intensity compared tofluorophore-tagged ubiquitin bound to tIVR. The percentage decrease ofthe fluorescence intensity of fluorescein, Alexa488, or Atto532-taggedubiquitin in the presence of tIVR compared to the absence of tIVR isgraphed (left). The percentage change of fluorescence intensity ofAlexa586, Atto532, and Alexa488-tagged chimeric polypeptide sensors withthree, two, or one ubiquitin binding domains upon binding free ubiquitinis graphed (right).

FIG. 15 provides a graph showing that Atto532-tISR L1 Cys2 shows a largefluorescence intensity change upon binding to ubiquitin. In thischimeric polypeptide sensor, the L1 Cys2, the linker between theIsoT^(ZnF) and the UIM, has a Cys conjugated with the fluorophore.Relative fluorescence intensity of Atto532-tISR L1 Cys2 is shown withdifferent concentrations of free ubiquitin proteins (triangles) orconjugated ubiquitin proteins.

FIG. 16 provides a graph showing that fluorescence intensity changeswith increasing amounts of free untagged ubiquitin. Fluorescenceintensities of emissions across 500 nm to 550 nm wavelengths of 3 nMfluorescein-tagged ubiquitin with 3 nM tIVR was measured. Concentrationsranging from 0 to 3000 nM of free untagged ubiquitin were added to themixture. Fluorescence intensity increased with higher concentrations offree ubiquitin. Further, there was no shift observed in the fluorescenceemission wavelength due to binding of free ubiquitin.

FIG. 17 provides a graph showing that anisotropy of fluorescein-taggedubiquitin proteins is changed when bound by tIVR. Anisotropy of mixtureswith 150 pM fluorescein-tagged ubiquitin proteins was measured andconcentrations of tIVR (0-10 nM) were added. Fluorescence was stimulatedwith polarized light at a 492 nm wavelength and polarized light emissionwas measured at 518 nm. Increasing the concentration of tIVR increasedthe anisotropy measured in the mixtures.

FIG. 18 provides a diagram of fluorophore-tagged ubiquitin proteinsbound and unbound to tIVR, and a graph showing that anisotropy offluorophore-tagged ubiquitin proteins decreases when they are releasedfrom tIVR. Anisotropy of a mixture of 20 nM fluoroscein-tagged ubiquitinproteins and 20 nM tIVR was measured. Adding free ubiquitin proteins tothe mixture displaced fluorophore-tagged ubiquitin proteins and reducedthe anisotropy measured from the mixture. The curve generated from thisexperiment can be used as a calibration standard in assays to measurefree ubiquitin proteins in complex biological samples.

FIG. 19 provides a graph showing that free ubiquitin proteins change theamount of quenching due to FRET between quencher-tagged tIVR andfluorophore-tagged ubiquitin proteins. When the tagged ubiquitin proteinis bound to the tIVR, the fluorescence of the tagged ubiquitin proteinis reduced by the quencher conjugated to the tIVR. Since free ubiquitinproteins disrupt the binding of the fluorophore-tagged ubiquitinproteins and tIVR tagged with the quencher, increasing levels of freeubiquitin proteins results in greater fluorescence intensity. Thischange in fluorescence depends not only on FRET, but also by thequenching induced by the interaction between tIVR and fluorophore-taggedubiquitin proteins. The curve generated from this experiment can be usedas a calibration standard in assays to measure free ubiquitin proteinsin complex biological samples.

FIG. 20 provides a graph showing that deubiquitinase enzyme activity canbe measured in a real-time assay. Release of ubiquitin by YUH1, adeubiquitinase enzyme, was measured as a fluorescence intensity change.Fluorescence intensity of a mixture containing YUH1, 500 nM ofubiquitin-D77 (i.e., ubiquitin conjugated to aspartic acid) assubstrate, 10 nM fluorescence-tagged ubiquitin, and 10 nM tIVR wasmeasured over time.

FIG. 21 provides a graph showing that deubiquitinase activity can bemonitored continuously. A real-time deubiquitinase assay usingAtto532-tagged ubiquitin proteins and tIVR is graphed. A mimic ofpolyubiquitinated-protein substrate, was digested with 25 nM Usp2cc or 3μM OTUB1 with or without UbcH5c in presence of 1 nM Atto532-taggedubiquitin protein and 1 nM tIVR to detect free ubiquitin released by thedeubiquitinases. OTUB1 activity is upregulated by UbcH5c.

FIG. 22 provides a graph showing that Atto532-tagged tIV L1 Cys binds tofree Nedd8 with a K_(d) of several μM.

FIG. 23 provides the construction scheme for the universal sensors. Achimeric polypeptide comprising a domain that binds to the hydrophobicpatch of ubiquitin protein and a domain that binds the surface aroundubiquitin are depicted. A linker connects these domains.

FIG. 24 provides a schematic illustration of the design concept for thechimeric polypeptide universal sensor for free ubiquitin. Ubiquitin isdepicted interacting with two binding domains that bind non-overlappingregions of ubiquitin (top). An example chimeric polypeptide comprisingtwo domains connected by two linkers is depicted binding to ubiquitin atthe two non-overlapping sites (bottom).

FIG. 25 provides a graph and table showing that the tSR chimeric peptideuniversal sensor does not discriminate among ubiquitin protein. Acompetition assay was performed where unconjugated free ubiquitinproteins, different kinds of conjugated free ubiquitin proteins, andNedd8 were added to mixtures of 5 nM Atto532-tagged ubiquitin proteinand 600 nM tSR. The relative fluorescence was measured. While tSR hadsimilar binding affinities for unconjugated ubiquitin proteins andconjugated ubiquitin proteins, tSR displayed specificity against Nedd8(top). The Ki values for unconjugated ubiquitin proteins and thedifferent kinds of conjugated free ubiquitin proteins are provided in atable (bottom).

FIG. 26 provides an illustration and graphs demonstrating that tSR canbe used for direct titration experiments. An illustration showsfluorophore-tagged tSR is shown binding to ubiquitin (left). Therelative fluorescence intensity changes of Alexa488-tagged tSR whenubiquitin is added is shown (middle). The relative fluorescence of 10 nMAlexa488 tSR when different concentrations of unconjugated ubiquitinprotein, conjugated ubiquitin protein, or Nedd8 are added (right).

DESCRIPTIONS OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the tIVR chimeric polypeptidesensor.

SEQ ID NO: 2 is the amino acid sequence of the tIVR L1 Cys chimericpolypeptide sensor.

SEQ ID NO: 3 is the amino acid sequence of the tIVR (R218C) chimericpolypeptide sensor.

SEQ ID NO: 4 is the amino acid sequence of the tIDR chimeric polypeptidesensor.

SEQ ID NO: 5 is the amino acid sequence of the tIV L1 Cys chimericpolypeptide sensor.

SEQ ID NO: 6 is the amino acid sequence of the tISR chimeric polypeptidesensor.

SEQ ID NO: 7 is the amino acid sequence of the tSR chimeric polypeptidesensor.

SEQ ID NO: 8 is the amino acid sequence of the IsoT^(ZnF) ubiquitinbinding domain.

SEQ ID NO: 9 is the amino acid sequence of the Vps27^(UIM) ubiquitinbinding domain.

SEQ ID NO: 10 is the amino acid sequence of the Ruz ubiquitin bindingdomain.

SEQ ID NO: 11 is the amino acid sequence of the Dsk^(UBA) ubiquitinbinding domain.

SEQ ID NO: 12 is the amino acid sequence of the S5a^(UIM) ubiquitinbinding domain.

SEQ ID NO: 13 is the amino acid sequence of the tIVR linker 1.

SEQ ID NO: 14 is the amino acid sequence of the tIVR linker 2.

SEQ ID NO: 15 is the amino acid sequence of the tIDR linker 1.

SEQ ID NO: 16 is the amino acid sequence of the tIDR linker 2.

SEQ ID NO: 17 is the amino acid sequence of the tIVR linker 1 with a Cysaddition.

SEQ ID NO: 18 is the amino acid sequence of the tIV linker 1 with a Cysaddition.

SEQ ID NO: 19 is the amino acid sequence of the tISR linker 1.

SEQ ID NO: 20 is the amino acid sequence of the tISR linker 2.

SEQ ID NO: 21 is the amino acid sequence of the tSR linker 1.

SEQ ID NO: 22 is the amino acid sequence of human ubiquitin protein.

SEQ ID NO: 23 is the amino acid sequence of the human Nedd8 protein.

SEQ ID NO: 24 is the amino acid sequence of the human SUMO1 protein.

SEQ ID NO: 25 is the amino acid sequence of the human SUMO2 protein.

SEQ ID NO: 26 is the amino acid sequence of the human SUMO3 protein.

SEQ ID NO: 27 is the amino acid sequence of ubiquitin (S20C).

SEQ ID NO: 28 is the amino acid sequence of a long tIVR linker 1.

SEQ ID NO: 29 is the amino acid sequence of OTUB1.

SEQ ID NO: 30 is the amino acid sequence of a short tIVR linker 1.

SEQ ID NO: 31 is the amino acid sequence of the IsoT^(ZnF) (S227C)ubiquitin binding domain.

SEQ ID NO: 32 is the amino acid sequence of Ub-GB1.

SEQ ID NO: 33 is the amino acid sequence of UBCH5C.

SEQ ID NO: 34 is the amino acid sequence of the DUIM ubiquitin bindingdomain.

SEQ ID NO: 35 is the amino acid sequence of the MIU ubiquitin bindingdomain.

SEQ ID NO: 36 is the amino acid sequence of the CUE ubiquitin bindingdomain.

SEQ ID NO: 37 is the amino acid sequence of the GAT ubiquitin bindingdomain.

SEQ ID NO: 38 is the amino acid sequence of the Jab1/MPN ubiquitinbinding domain.

SEQ ID NO: 39 is the amino acid sequence of the NZF ubiquitin bindingdomain.

SEQ ID NO: 40 is the amino acid sequence of the UBZ ubiquitin bindingdomain.

SEQ ID NO: 41 is the amino acid sequence of the UBS ubiquitin bindingdomain.

SEQ ID NO: 42 is the amino acid sequence of the UBM ubiquitin bindingdomain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new chimeric polypeptides thatselectively bind to ubiquitin proteins or ubiquitin-like proteins, andnew methods to detect amounts of ubiquitin proteins or ubiquitin-likeproteins. These methods can be tailored to detect a distinct targetubiquitin proteins or ubiquitin-like proteins, including total ubiquitinproteins, free ubiquitin proteins, conjugated ubiquitin proteins, totalubiquitin-like proteins, free ubiquitin-like proteins, or conjugatedubiquitin proteins. The methods also include a novel deubiquitinaseassay. Further, the present invention can be utilized to screen foragents that alter ubiquitin protein or ubiquitin-like proteinconjugation.

In certain embodiments, the chimeric polypeptides (also may also bereferred to as “sensors”) include two or more binding domains that bindto a ubiquitin protein or a ubiquitin-like protein. The binding domainsare connected by one or more linker(s). In particular embodiments, thebinding domains of a chimeric polypeptide bind to non-overlappingregions within a single ubiquitin protein or ubiquitin-like polypeptide,and the binding domains can bind the ubiquitin protein or ubiquitin-likeprotein simultaneously. This results in the chimer polypeptide havinggreater binding affinity and specificity than any of the binding domainsalone.

In related embodiments, the present invention provides new methods todetect and measure amounts of free ubiquitin proteins, freeubiquitin-like proteins, conjugated ubiquitin proteins, or conjugatedubiquitin-like proteins. In various embodiments, the chimericpolypeptides of the present invention can be used in direct titrationassays, competitive binding assays, fluorescence resonance energytransfer (FRET), and deubiquitination assays to determine an amount of afree ubiquitin protein, a free ubiquitin-like protein, a conjugatedubiquitin protein, or a conjugated ubiquitin-like protein. The methodsand assays of the present invention may also be used to determine thetotal amount of a ubiquitin protein or a ubiquitin-like protein, as wellas the percentage the total ubiquitin protein that is free orconjugated. The present invention allows for the detection of freeubiquitin proteins, free ubiquitin-like proteins, conjugated ubiquitinproteins, or conjugated ubiquitin-like proteins by measurement ofdetectable signals, by means including fluorescence intensity orfluorescence anisotropy. The chimeric polypeptides can be used to detectubiquitin protein or ubiquitin-like protein in vitro, as well inbiological samples, including extracts from cell cultures or tissues.

The present invention has several advantages. Despite the importance andabundance of the ubiquitin system, few methods exist for detecting anamount of free ubiquitin protein or ubiquitin-like protein ordetermining the amount of a total ubiquitin protein or ubiquitin-likeprotein that is in free or conjugated. Methods that do exist can beinaccessible to many laboratories. In one embodiment, the presentinvention provides novel methods of detecting free ubiquitin proteins orfree ubiquitin-like proteins, and it does so without the use of highperformance liquid chromatography, mass spectrometry, heavy-isotopestandards, antibodies, film, or gel electrophoresis. Furthermore, onlyone other method has been described that is able to specifically measureubiquitin that has an intact free C-terminus, but unlike the presentinvention, that method is unsuitable for complex biological samples suchas cell or tissue lysates, requires use of radioactive ATP, and is notamenable to high-throughput assays. The present invention provides novelchimeric polypeptides that bind to ubiquitin proteins or ubiquitin-likeproteins with high affinity and specificity. These chimeric polypeptidescan be used to measure ubiquitin protein or ubiquitin-like protein invitro, and can be used with complex cell or tissue extracts. The methodsof the present invention can also be used in assays to identify orevaluate proteasome inhibitors, deubiquitinating enzymes(deubiquitinases, or “DUBs”), and DUB inhibitors, or other ubiquitinpathway modulators (e.g., agonists or inhibitors) for their effects onthe amount of free ubiquitin. The present invention also can be used inquantitative, real-time assays of DUB activities that, unlike existingassay formats, can use any form of conjugated ubiquitin as thesubstrate.

I. Ubiquitin Sensors

The present invention provides chimeric polypeptides, i.e., chimericpolypeptide sensors, that comprise two or more sequences that bind to aubiquitin protein or a ubiquitin-like protein, and methods comprisingthe use of the chimeric polypeptide sensors to detect an amount of aubiquitin protein or a ubiquitin-like protein in a sample or mixture. Inone aspect, the present invention comprises a chimeric polypeptidesensor comprising two or more polypeptide sequences that bind aubiquitin protein or a ubiquitin-like protein wherein the sequences bindnon-overlapping regions of the ubiquitin protein or ubiquitin likeprotein; and one or more linker(s), wherein the linker(s) connect thetwo or more sequences.

In certain embodiments, the chimeric polypeptide sensors bind to theubiquitin proteins or the ubiquitin-like proteins in both their “free”state, (i.e., not conjugated to a non-ubiquitin protein) and in their“conjugated” state (i.e., conjugated to a non-ubiquitin protein), e.g.,with approximately the same or similar affinity. As used herein, “free”ubiquitin proteins and “free” ubiquitin-like proteins include ubiquitinprotein monomers and ubiquitin-like protein monomers, as well as“polyubiquitins” and “polyubiquitin-like proteins,” which comprise orconsist of two or more ubiquitins or two or more ubiquitin-likeproteins, respectively. “Total” ubiquitin protein refers to combinedfree ubiquitin protein and conjugated ubiquitin protein. “Total”ubiquitin-like protein refers to combined free ubiquitin-like proteinand conjugated ubiquitin-like protein. In some embodiments, the chimericpolypeptide sensors preferentially bind to a free ubiquitin protein or afree ubiquitin-like protein as compared to the corresponding conjugatedubiquitin protein or conjugated ubiquitin-like protein (i.e., the sameprotein conjugated to a non-ubiquitin or non-ubiquitin-like protein),e.g., with an affinity at least 1.5-fold greater, 2.0-fold greater,3.0-fold greater, at least 4.0-fold greater, at least 5.0-fold greater,at least 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 50-fold greater, or at least 100-fold greater.

In some embodiments, the chimeric polypeptide sensor preferentiallybinds to the target ubiquitin protein or the target ubiquitin-likeprotein as compared to another ubiquitin protein or ubiquitin-likeprotein. In certain embodiments, a chimeric polypeptide has an affinityfor a target protein at least 1.5-fold greater, 2.0-fold greater,3.0-fold greater, at least 4.0-fold greater, at least 5.0-fold greater,at least 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 50-fold greater, or at least 100-fold greater than itsaffinity for another ubiquitin or ubiquitin-like protein. In someembodiments, the chimeric polypeptide sensor preferentially binds to aubiquitin protein as compared to a ubiquitin-like protein. In someembodiments, the chimeric polypeptide sensor preferentially binds tototal ubiquitin protein as compared to total ubiquitin-like protein. Inother embodiments, the chimeric polypeptide sensor preferentially bindsto a ubiquitin-like protein as compared to a ubiquitin protein. In someembodiments, the chimeric polypeptide sensor preferentially binds tototal ubiquitin-like protein as compared to total ubiquitin protein.

In some embodiments, the chimeric polypeptide sensor preferentiallybinds to a free ubiquitin as compared to the conjugated ubiquitin. Insome embodiments, the chimeric polypeptide sensor preferentially bindsto a free ubiquitin-like protein as compared to the conjugatedubiquitin-like protein. In some embodiments, the chimeric polypeptidesensor preferentially binds to free ubiquitin-like protein compared toconjugated ubiquitin-like proteins and ubiquitin proteins. It will beunderstood by those in the art that preferential binding to a particularubiquitin protein or a particular ubiquitin-like protein meanspreferential binding compared to other proteins that are not theparticular ubiquitin protein or the particular ubiquitin-like protein.

The term “polypeptide” as used herein interchangeably with the term“protein” describes linear molecular chains of amino acids, includingsingle chain proteins or their fragments, containing multiple aminoacids. Furthermore, peptidomimetics of such proteins/polypeptides whereamino acid(s) and/or peptide bond(s) have been replaced by functionalanalogues are also encompassed by the invention. Such functionalanalogues include all known amino acids other than the 20 gene-encodedamino acids, such as selenocysteine. The terms “polypeptide” and“protein” also refer to naturally modified polypeptides/proteins wherethe modification is effected e.g. by glycosylation, acetylation,phosphorylation and similar modifications which are well known in theart. The terms “terminal” and “terminus” are well understood in the artand are used here interchangeably.

The recitations “sequence identity”, “percent identity”, “percenthomology”, or, for example, comprising a “sequence 50% identical to,” asused herein, refer to the extent that sequences are identical on anucleotide-by-nucleotide basis or an amino acid-by-amino acid basis overa window of comparison. Thus, a “percentage of sequence identity” may becalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, I) or the identical aminoacid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr,Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity.

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) can be performedas follows. To determine the percent identity of two amino acidsequences, or of two nucleic acid sequences, the sequences can bealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In certain embodiments, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch, (1970, J.Mol. Biol. 48: 444-453) algorithm which has been incorporated into theGAP program in the GCG software package, using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package, using anNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. Another exemplary set ofparameters includes a Blossum 62 scoring matrix with a gap penalty of12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Thepercent identity between two amino acid or nucleotide sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (1989,Cabios, 4: 11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The peptide sequences described herein can be used as a “query sequence”to perform a search against public databases to, for example, identifyother family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.

In certain embodiments, the invention comprises a chimeric polypeptidesensor that binds to a free ubiquitin protein with high affinity andselectivity compared to total ubiquitin protein and compared toubiquitin-like proteins. In some embodiments, the chimeric polypeptidecomprises more than one ubiquitin binding domain, e.g., two or threeubiquitin binding domains, wherein the binding domains bind tonon-overlapping regions of the ubiquitin protein. In some embodiments,the chimeric polypeptide sensor binds to the ubiquitin protein withhigher affinity and specificity than a polypeptide comprising a singlebinding domain.

In certain embodiments, the invention comprises a chimeric polypeptidesensor that binds to free ubiquitin-like protein with high affinity andspecificity. In some embodiments, the chimeric peptide sensor comprisesmore than one ubiquitin-like protein binding domain, e.g., two or threeubiquitin-like protein binding domains, wherein the binding domains bindto non-overlapping regions of the ubiquitin-like protein. In someembodiments, the chimeric polypeptide sensor binds to the ubiquitin-likeprotein with higher affinity and specificity than a polypeptidecomprising a single binding domain.

In particular embodiments, the ubiquitin protein binding domains andubiquitin-like protein binding domains are polypeptide sequences thatbind to the ubiquitin protein or the ubiquitin-like protein, includingany of those described herein.

In particular embodiments, a chimeric polypeptide of the presentinvention comprises or consists of two polypeptide sequences that bindnon-overlapping regions of the same ubiquitin protein or ubiquitin-likeprotein and one or more linker moiety. In one embodiment, the chimericpolypeptide includes one linker moiety that connects the two polypeptidesequences.

In particular embodiments, a chimeric polypeptide of the presentinvention comprises or consists of three polypeptide sequences that bindnon-overlapping regions of the same ubiquitin protein or ubiquitin-likeprotein and one, two or more linker moieties. In one embodiment, thechimeric polypeptide includes one linker moiety that connects all threepolypeptide sequences. For example, the linker may be a linker capableof binding to three polypeptide sequences. In one embodiment, thechimeric polypeptide includes two linker moieties, each of whichconnects two of the polypeptide sequences.

In some embodiments, the invention comprises a chimeric polypeptide thatbinds to a ubiquitin protein or a ubiquitin-like protein, comprising inthe following order: a first polypeptide sequence that binds to a firstregion of the ubiquitin protein or the ubiquitin-like protein; a firstlinker that connects the first polypeptide sequence to a secondpolypeptide sequence that binds to a second region of the ubiquitinprotein or the ubiquitin-like protein, a second linker that connects thesecond polypeptide sequence to a third polypeptide sequence that bindsto a third region of the ubiquitin protein or the ubiquitin-likeprotein, whereas the first, second and third regions of the ubiquitinprotein or the ubiquitin-like protein bound by the first, second, andthird polypeptide sequences are non-overlapping. In some embodiments ofthe invention, the chimeric polypeptide sensor comprises more than threeubiquitin protein or ubiquitin-like protein binding domains that bind tonon-overlapping regions of the ubiquitin protein or the ubiquitin-likeprotein.

Nucleotide and amino acid sequences of ubiquitin proteins are known inthe art. In certain embodiments, a ubiquitin protein according to theinvention has at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identity to a known ubiquitin protein, suchas the human ubiquitin protein. In some embodiments, the ubiquitinprotein is the human ubiquitin protein. In other embodiments, theubiquitin according to the invention includes naturally occurring orengineered variants of ubiquitin. The amino acid sequence of the humanubiquitin protein is provided in SEQ ID NO: 22.

Ubiquitin proteins also include naturally occurring alleles and humanengineered variants of a known ubiquitin protein. In some embodiments, aubiquitin protein comprises the sequence of a known ubiquitin protein,but with one or more amino acid deletion, addition, or substitution, forexample, for the purposes of generating a site on the ubiquitin proteinto conjugate a detectable label. In some embodiments, the ubiquitinprotein comprises a substitution of the serine at amino acid position 20with a cysteine (S20C) to allow for covalent attachment of a detectablelabel to the ubiquitin protein SEQ ID NO 27].

In certain embodiments, a ubiquitin binding domain, according to theinvention, has at least 75, least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% identity, or 100% amino acidsequence identity to a known ubiquitin binding domain. Ubiquitin bindingdomains refer to a diverse family of dissimilar protein modules thatbind ubiquitin. Many ubiquitin binding domains have been described inthe art, and they include at least 19 different binding domains. Theseubiquitin binding domains include, but are not limited to, the UBA(Ubiquitin Associated domain), UIM (Ubiquitin Interacting Motif), MIU(Motif Interacting with Ubiquitin; SEQ ID NO: 35) domain, DUIM(double-sided ubiquitin-interacting motif, SEQ ID NO: 34), CUE (couplingof ubiquitin conjugation to ER degradation; SEQ ID NO: 36) domain, NZF(Np14 zinc finger; SEQ ID NO: 39), A20 ZnF (zinc finger), Ruz (RABEX-5zinc finger), UBP ZnF or BUZ (ubiquitin-specific processing proteasezinc finger), UBZ (ubiquitin-binding zinc finger; SEQ ID NO: 40), UEV(ubiquitin-conjugating enzyme E2 variant), PFU (PLAA family ubiquitinbinding), GLUE (GRAM-like ubiquitin binding in EAP45), GAT(Golgi-localized, Gamma-ear-containing, Arf-binding; SEQ ID NO: 37),Jab1/MPN (Jun kinase activation domain binding/Mpr1p and Pad1pN-termini; SEQ ID NO: 38), WD40 βPrps (WD40 β-propellers), UBM(Ubiquitin binding motif; SEQ ID NO: 42), UBS (Ubiquitin bindingsurface; SEQ ID NO: 41), UBR (ubiquitin binding region), and Ubc(ubiquitin-conjugating enzyme). UBDs are structural motifs, many asshort as 20-40 amino acids long, with low sequence conservation, thatare found in all eukaryotes. These binding domains bind to specificregions of ubiquitin.

In some embodiments of the present invention, the chimeric polypeptidesensor comprises a ubiquitin binding domain that binds to the ubiquitinC-terminus. In some embodiments, the chimeric polypeptide sensorcomprises a ubiquitin binding domain that binds to the surfacehydrophobic patch of a ubiquitin protein. In other embodiments, thechimeric polypeptide comprises a ubiquitin binding domain that binds tothe ubiquitin protein at the surface near Asp58.

The ubiquitin C-terminus is known in the art as a site of attachmentwhere a ubiquitin protein can be conjugated to a non-ubiquitin protein;thus, this C-terminus is exposed in free ubiquitin. When a ubiquitinprotein is conjugated to a non-ubiquitin protein at the ubiquitin Cterminus, the non-ubiquitin protein is referred to in the art as being“ubiquitinated”. Without wishing to be bound to any particular theory,it is believed that chimeric polypeptide sensors that include a sequencethat binds to a ubiquitin C-terminal domain have greater bindingaffinity for a free ubiquitin protein (including free polyubiquitin)than for a conjugated ubiquitin protein. Known ubiquitin binding domainsthat can bind ubiquitin protein at the ubiquitin C-terminus include ZnFUBP and Ubc. Accordingly, in certain embodiments, chimeric polypeptidesensors of the present invention comprise a sequence that binds to aubiquitin C-terminal domain, and these sensors preferentially bind to afree ubiquitin protein as compared to the same ubiquitin protein when itis conjugated. In certain embodiments, these chimeric polypeptidesensors bind to the free ubiquitin protein with at least 1.5-fold,two-fold, three-fold, four-fold, five-fold or ten-fold greater affinitythan they bind to the conjugated ubiquitin protein.

The surface near the ubiquitin Asp58 amino acid residue is a region ofubiquitin protein that can interact with proteins containing certainubiquitin binding domains. Known ubiquitin binding domains that can bindubiquitin protein at the surface near the ubiquitin Asp58 include Ruz.

The ubiquitin hydrophobic patch is a region of ubiquitin that caninteract with proteins containing certain ubiquitin binding domains. Theubiquitin hydrophobic patch is located on the ubiquitin protein near theubiquitin Ile44 residue. Typically, ubiquitin binding domains thatcomprise an alpha helix can interact with mono-ubiquitin proteins at theubiquitin hydrophobic patch. Known ubiquitin binding domains that canbind ubiquitin protein at the ubiquitin hydrophobic patch include UBA,UIM, DUIM, MIU, CUE, GAT, Jab1/MPN, NZF, UBZ, UBS, and UBM.

In some embodiments, the invention comprises a chimeric polypeptidecomprising two or more polypeptide sequences that bind a ubiquitinprotein or a ubiquitin-like protein, wherein the sequences bindnon-overlapping regions of the ubiquitin protein or ubiquitin-likeprotein, and one or more linker(s), wherein the linker(s) connects thetwo or more polypeptide sequences. In some embodiments, the inventioncomprises a chimeric polypeptide wherein the sequences comprise asequence that binds to the ubiquitin C terminus and a sequence thatbinds to the ubiquitin hydrophobic patch. In some embodiments, theinvention comprises a chimeric polypeptide wherein the sequencescomprise a sequence that binds to the ubiquitin C terminus and asequence that binds the surface around Asp58 of ubiquitin. In someembodiments, the invention comprises a chimeric polypeptide, wherein thesequences comprise a sequence that binds to the ubiquitin hydrophobicpatch and a sequence that binds to surface around Asp58 of ubiquitin. Insome embodiments, the invention comprises a chimeric polypeptidecomprising a sequence that binds to the ubiquitin C terminus, a sequencethat binds to the ubiquitin hydrophobic patch, and a sequence that bindsto the surface around Asp58 of ubiquitin.

In some embodiments of the invention, a chimeric polypeptide comprisinga ubiquitin binding domain that binds to a ubiquitin protein at aparticular binding site will have similar binding affinities andspecificities (e.g., with respect to targets of ubiquitin protein) asanother chimeric polypeptide sensor comprising a different ubiquitinbinding region that binds to the same binding site of the ubiquitinprotein. Thus, in certain embodiments of the invention, any of thespecific ubiquitin binding domains that bind to sites on the ubiquitinprotein are interchangeable with other ubiquitin binding domains fromother proteins that contain the same kind of ubiquitin binding domain(e.g., binds to the same site on the ubiquitin protein), and ubiquitinbinding domains that bind to the same sites on the ubiquitin protein maybe swapped to achieve a chimeric polypeptide sensor with similar bindingaffinity and selectivity to a target ubiquitin protein. Further, incertain embodiments of the invention, the specific ubiquitin bindingdomains that bind to sites on the ubiquitin protein are interchangeablewith other ubiquitin binding domains that bind to ubiquitin at the samesites on the ubiquitin protein, and may be interchanged to achieve achimeric polypeptide sensor with similar binding affinity andselectivity to a target ubiquitin protein. The same concept holds truefor chimeric polypeptide sensors that bind to a ubiquitin-like protein.Thus, the present invention is not restricted to specific polypeptidedomains that bind to a ubiquitin protein or a ubiquitin-like protein onmultiple sites, but rather, the present invention encompasses chimericpolypeptide sensors that comprise any ubiquitin binding domains thatbind the ubiquitin or ubiquitin-like protein on the same sites.

In some embodiments in the present invention, the chimeric polypeptidecomprises an order, from N-terminal to C-terminal, of an arrangement ofnon-overlapping ubiquitin protein binding domains or ubiquitin-likeprotein binding domains. In some embodiments, the order of the bindingdomains, from N-terminal to C-terminal is interchangeable. Thus, in someembodiments, a chimeric polypeptide comprising an order of a first, asecond, and a third binding domain will have similar binding affinitiesand specificities for target ubiquitin proteins or ubiquitin-likeproteins as a chimeric polypeptide sensor comprising an order of thethird, the second, and the first binding domain, and will have similarbinding affinities and specificities as a chimeric polypeptide sensorcomprising an order of the second, the third, and the first bindingdomain, and will have the similar binding affinities as a chimericpolypeptide sensor comprising an order of the first, the third, and thesecond binding domain. Thus, in some embodiments, properties of thechimeric polypeptide sensor do not depend on the order the ubiquitin orubiquitin-like protein binding domains are arranged. Further, in someembodiments of the invention, the properties of a chimeric polypeptidesensor that binds to a ubiquitin protein or a ubiquitin-like proteinwith high affinity comprising two or more ubiquitin protein orubiquitin-like protein binding domains does not depend on the order theubiquitin protein or ubiquitin-like protein binding domains arearranged.

In certain embodiments of the invention, a linker moiety (or “linker”)is a moiety that connects two ubiquitin protein or ubiquitin-likeprotein binding domains. In some embodiments, the linker must be anappropriate length to allow the binding domains to simultaneously bindto the same ubiquitin protein or ubiquitin-like protein. In certainembodiments, a linker moiety is a chemical moiety or a polypeptide. Avariety of linker moieties are known and available in the art. Incertain embodiments, linkers comprise at least one amino acid. In someembodiments, linkers comprise more than two amino acids. In someembodiments, linkers comprise more than three amino acids, more thanfour amino acids, more than five amino acids, more that ten amino acids,more than 20 amino acids, or more than 30 amino acids. In someembodiments, linkers comprise between two and 30 amino acids. In someembodiments, linkers comprise between three and 30 amino acids, betweenthree and 15 amino acids, between three and twelve amino acids, betweenfour and twelve amino acids, between five and twelve amino acids,between five and ten amino acids, between five and 15 amino acids,between four and 30 amino acids, between five and 30 amino acids,between ten and 30 amino acids, between 20 and 30 amino acids, orbetween 10 and 50 amino acids. In certain embodiments, a linker includesless than or equal to 100, 50, 40, 30, 20, 15, twelve, ten, nine, eight,seven, six, five, four, three, or two amino acid residues. In someembodiments, linkers are comprised of or consist of one or more aminoacid(s) selected from the group of glycine, serine, or alanine residues.In some embodiments, linkers further comprise a cysteine residue. Insome embodiments, linkers are comprised of or consist of one or moreamino acid(s) selected from the group of glycine, serine, alanine andcysteine residues. In some embodiments, the cysteine residue isconjugated to a detectable label. In particular embodiments, a linkermoiety comprises or consists of any of the amino acid sequences setforth in SEQ ID NOs: 13, 15, 16, 17, 19, and 28. Examples ofillustrative linkers and polypeptide sensors of the present inventionare also provided in the accompanying Examples. In some embodiments, thebinding affinity of the chimeric polypeptide sensor to a targetubiquitin protein or ubiquitin-like protein is influenced by length ofthe linker(s). One of skill in the art will appreciate that a linkerconnecting two or more ubiquitin binding domains should be of sufficientlength and flexibility to allow the ubiquitin binding domains to contactand bind the regions of the ubiquitin protein that they bind, whilethese ubiquitin binding domains are connected via the linker.Accordingly, the length of the linker may be determined based, in part,on knowledge of the regions of the ubiquitin protein (or ubiquitin-likeprotein) bound by the two binding domains connected via the linker.

In some embodiments, the invention comprises a chimeric polypeptidesensor that comprises or consists of two or more ubiquitin orubiquitin-like protein binding domains and at least one linker.

In some embodiments, the invention comprises a chimeric polypeptidewherein the sequences comprise the ZnF UBP binding domain fromIsopeptidase T. In some embodiments, the invention comprises a chimericpolypeptide wherein the sequences comprise the Ruz domain from Rabex-5.In some embodiments, the invention comprises a chimeric polypeptidewherein the sequences comprise the ubiquitin interacting motif (UIM)from Vps27. In some embodiments, the invention comprises a chimericpolypeptide wherein the sequences comprise the ubiquitin associated(UBA) domain from Dsk2.

In some embodiments, the invention comprises a chimeric polypeptidesensor, wherein the sequences comprise or consist of the ZnF UBP bindingdomain from Isopeptidase T and the Ruz domain from Rabex-5. In someembodiments, the invention comprises a chimeric polypeptide sensor,wherein the sequences comprise or consist of the ZnF UBP binding domainfrom Isopeptidase T and the ubiquitin interacting motif from Vps27. Insome embodiments, the invention comprises a chimeric polypeptide,wherein the sequences comprise or consist of the Ruz domain from Rabex-5and the ubiquitin interacting motif from Vps27. In some embodiments, theinvention comprises a chimeric polypeptide sensor, wherein the sequencescomprise or consist of the ZnF UBP binding domain from Isopeptidase T,the Ruz domain from Rabex-5, and the ubiquitin interacting motif fromVps27.

In some embodiments, the invention comprises a chimeric polypeptidesensor that comprises or consists of two or more ubiquitin orubiquitin-like protein binding domains and at least one linker, whereinthe chimeric polypeptide sensor does not comprise a binding domain thatbinds the ubiquitin protein or ubiquitin-like protein at its C-terminusor C-terminal domain. In certain embodiments, such a chimericpolypeptide sensor binds with approximately the same affinity and/orspecificity to a free ubiquitin protein or free ubiquitin-like proteinas compared to the same ubiquitin protein or ubiquitin-like protein whenconjugated.

In some embodiments of the invention, a ZnF UBP binding domain fromIsopeptidase T according to the invention has at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identity to a known zinc finger binding domain from Isopeptidase Tprotein, such as the from the human Isopeptidase T protein. In someembodiments of the invention, a zinc finger binding domain fromIsopeptidase T has at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8.

In some embodiments, a Ruz domain from Rabex-5, according to theinvention has at least 80%, at least 85%, least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity to a known Ruz domain fromRabex-5, such as from the human Rabex-5 protein. In some embodiments ofthe invention, a Ruz domain from Rabex-5 has at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, at least 99%, or 100% identityto SEQ ID NO: 10.

In some embodiments, a ubiquitin interacting motif from Vps27 accordingto the invention has at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100% identity to a known ubiquitininteracting motif from Vps27, such as from a yeast Vps27 protein. Insome embodiments of the invention, a ubiquitin interacting motif fromVps2 has at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

In some embodiments, a ubiquitin associated domain from Dsk2 accordingto the invention has at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100% identity to a known ubiquitinassociated domain from Dsk2, such as a yeast Dsk2 protein. In someembodiments of the invention, ubiquitin associated domain from Dsk2 hasat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% identity to SEQ ID NO: 11.

In some embodiments, ubiquitin interacting motif from the S5a subunit ofthe proteasome according to the invention has at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identity to a known ubiquitin interacting motif from the S5a subunit ofthe proteasome, such as the human S5a protein. In some embodiments ofthe invention, a ubiquitin interacting motif from the S5a subunit of theproteasome has at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99% or 100% identity to SEQ ID NO: 12.

In some embodiments, the invention comprises a chimeric polypeptidesensor comprising the ZnF UBP binding domain from Isopeptidase T, theRuz domain from Rabex-5, the ubiquitin interacting motif from Vps27, anda first polypeptide linker and a second polypeptide linker. In someembodiments, the chimeric polypeptide sensor comprises the sequence ofSEQ ID NO: 1 and is termed tIVR. In some embodiments, tIVR has a highbinding affinity and specificity for free ubiquitin, as compared toconjugated ubiquitin and compared to ubiquitin-like proteins. In someembodiments, tIVR is tagged with a detectable label. In someembodiments, tIVR is tagged with a fluorophore. In some embodiments, thetIVR is tagged with a fluorophore at R218C (SEQ ID NO:3). In someembodiments, the tIVR is tagged with a fluorophore at the firstpolypeptide linker. In some embodiments, the tIVR is tagged with afluorophore at the first polypeptide linker and comprises the sequenceSEQ IS NO: 2. In some embodiments, the tIVR has at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 1.

In some embodiments, the invention comprises a chimeric polypeptidesensor comprising the ZnF UBP binding domain from Isopeptidase T and theubiquitin associated domain from Dsk2. In some embodiments, the chimericpolypeptide sensor comprises the Ruz domain from Rabex-5 and theubiquitin associated domain from Dsk2. In some embodiments, the chimericpolypeptide sensor comprises the ZnF UBP binding domain fromIsopeptidase T, the Ruz domain from Rabex-5, and the ubiquitinassociated domain from Dsk2. In some embodiments, the chimericpolypeptide sensor comprises the sequence of SEQ ID NO: 4. and is termedtIDR. In some embodiments, tIDR has a high binding affinity andspecificity for free ubiquitin protein, as compared to conjugatedubiquitin protein and compared to ubiquitin-like protein. In someembodiments, tIDR is tagged with a detectable label. In someembodiments, tIDR is tagged with a fluorophore. In some embodiments, thetIDR has at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity to SEQ ID NO: 4.

In some embodiments, the invention comprises a chimeric polypeptidesensor comprising the ZnF UBP binding domain from Isopeptidase T, theRuz domain from Rabex-5, the ubiquitin interacting motif from S5a, and afirst polypeptide linker and a second polypeptide linker. In someembodiments, the chimeric polypeptide sensor comprises the sequence ofSEQ ID NO:6 and is termed tISR. In some embodiments, tISR has a highbinding affinity and specificity for free ubiquitin, as compared toconjugated ubiquitin and compared to ubiquitin-like proteins. In someembodiments, tISR is tagged with a detectable label. In someembodiments, tISR is tagged with a fluorophore. In some embodiments, thetISR is tagged with a fluorophore on a cysteine on linker 2. In someembodiments, the tISR is tagged with a fluorophore at the secondpolypeptide linker and comprises the sequence SEQ ID NO: 6. In someembodiments, the tISR has at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6.

In some embodiments, the invention comprises a chimeric polypeptidesensor comprising the Ruz domain from Rabex-5 and the ubiquitininteracting motif from the S5a subunit of the proteasome. In someembodiments, the chimeric polypeptide sensor comprises the sequence ofSEQ ID NO: 7. wherein the chimeric polypeptide sensor is termed tSR. Insome embodiments, the tSR has a high binding affinity and specificityfor total ubiquitin protein, compared to ubiquitin-like proteins. Insome embodiments, the tSR has similar binding affinities to conjugatedubiquitin protein and free ubiquitin protein. In some embodiments, tSRis tagged with a detectable label. In some embodiments, tSR is taggedwith a fluorophore. In some embodiments, the tSR has at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% identity to SEQ ID NO: 7.

In some embodiments, the invention comprises a chimeric polypeptidewherein the sequences bind to non-overlapping regions of aubiquitin-like protein. In some embodiments, the invention comprises achimeric polypeptide wherein the sequences bind to non-overlappingregions of Nedd8. In some embodiments, the invention comprises achimeric polypeptide wherein the sequences bind to non-overlappingregions of SUMO.

In some embodiments, a ubiquitin-like protein according to the inventionis a protein having properties that are similar to those of ubiquitin.Like ubiquitin, other members of the ubiquitin-protein family can beconjugated to non-ubiquitin-like proteins, and can formpolyubiquitin-like protein chains comprising non-ubiquitin proteins.Thus, like ubiquitin, other members of the ubiquitin-like protein familycan be free or conjugated. Members of the ubiquitin-like protein familyare well known in the art, and regulate diverse biological processes.Examples of ubiquitin-like proteins include Nedd8, SUMO-1, SUMO-2,SUMO-3, NUB1, PIC1, UBL3, UBL5, FAT10 and ISG15.

As used herein, the terms “Nedd8” and “neuronal precursor cell expresseddevelopmentally down-regulated protein 8” refer to a member of thefamily of ubiquitin-like proteins that is covalently attached to targetproteins. The human, mouse, and rat Nedd8 sequences are each 81 aminoacids in length and are about 6 kDa. The terms “Nedd8” and “neuronalprecursor cell expressed developmentally down-regulated protein 8” alsorefer to the yeast Rubl protein. Nucleotide and amino acid sequences ofNEDD8 proteins are known in the art.

In certain embodiments, a Nedd8 protein according to the invention hasat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% identity to a known Nedd8 protein, such as the humanNedd8 protein. In some embodiments, the Nedd8 protein has at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100% identity to the human Nedd8 protein SEQ ID NO: 23. In someembodiments, the Nedd8 protein is the human Nedd8 protein. In otherembodiments, the Nedd8 protein according to the invention includesnaturally occurring or engineered variants of Nedd8. Also encompassed by“Nedd8” are naturally occurring alleles and human engineered variants ofa known Nedd8 protein. In some embodiments, Nedd8 comprises the sequencein SEQ ID NO: 23 with an amino acid substitution, for example for thepurposes of generating a site on the Nedd8 protein to conjugate adetectable label.

In some embodiments, the invention comprises a chimeric polypeptidesensor comprising two or more polypeptide sequences that bind a Nedd8protein wherein the sequences bind non-overlapping regions of the Nedd8protein; and one or more linkers, wherein the linkers connect two ormore of the sequences. In some embodiments, the invention comprises thechimeric polypeptide sensor wherein the chimeric polypeptide has threepolypeptide sequences that bind to non-overlapping regions of Nedd8proteins connected by two linkers. In some embodiments, the chimericpolypeptide sensor has a selective binding affinity for free Nedd8protein compared to conjugated Nedd8 protein. In some embodiments, thechimeric polypeptide sensor has a selective binding affinity conjugatedNedd8 protein compared to free Nedd8 protein. In some embodiments, thechimeric polypeptide sensor has a selective binding affinity for Nedd8compared to ubiquitin proteins or other ubiquitin-like proteins.

In certain embodiments, the invention comprises a chimeric polypeptidesensor comprising the ZnF UBP binding domain from Isopeptidase T and theubiquitin interacting motif from Vps27. In some embodiments, chimericpolypeptide sensor comprising the zinc finger binding domain fromIsopeptidase T and the ubiquitin interacting motif from Vps27 comprisesthe sequence listed in SEQ ID NO: 5. wherein the chimeric polypeptidesensor is termed tIV. In some embodiments, the tIV comprises a selectivebinding affinity for free Nedd8 compared to conjugated Nedd8. In someembodiments, tIV is tagged with a fluorophore. In some embodiments, tIVis tagged with a fluorophore connected at a linker. In some embodiments,the tIV has at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity to SEQ ID NO: 5.

Small ubiquitin-like modifier (SUMO) proteins are a group of smallproteins that bind lysine residues of target proteins and thereby modifytarget protein activity, stability, and sub-cellular localization. SUMO2and SUMO3 proteins share a high degree of similarity (95% sequenceidentity), but are relatively distinct from SUMO1 (only 50% sequenceidentity). SUMO conjugation (or “sumoylation”) is a highly volatileprocess, with various enzymes involved in the conjugation. A largeportion of SUMO conjugation targets are transcription factors and othernuclear proteins involved in gene expression.

In certain embodiments of the invention, a SUMO protein according to theinvention has at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% identity to a known SUMO protein, suchas the human SUMO1, SUMO2, or SUMO3 proteins. In some embodiments, theSUMO protein has at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100% identity to the human SUMO proteinsset forth in SEQ ID NO: 24-26. In some embodiments, the SUMO protein isone of the human SUMO proteins. In other embodiments, the SUMO proteinaccording to the invention includes naturally occurring or engineeredvariants of SUMO. Also encompassed by “SUMO” are naturally occurringalleles and human engineered variants of a known SUMO protein. In someembodiments, SUMO comprises a sequence set forth in SEQ ID NO: 24-26with an amino acid substitution, for example for the purposes ofgenerating a site on the SUMO protein to conjugate a detectable label.

In some embodiments, the invention comprises a chimeric polypeptidecomprising two or more polypeptide sequences that bind a SUMO proteinwherein the sequences bind non-overlapping regions of the SUMO proteins;and one or more linkers, wherein the linkers connect two or more of thesequences. In some embodiments, the invention comprises the chimericpolypeptide wherein the chimeric polypeptide has three polypeptidesequences that bind to non-overlapping regions of SUMO proteinsconnected by two linkers. In some embodiments, the chimeric polypeptidesensor has a selective binding affinity for free SUMO protein comparedto conjugated SUMO protein. In some embodiments, the chimericpolypeptide sensor has a selective binding affinity conjugated SUMOprotein compared to free SUMO protein. In some embodiments, the chimericpolypeptide sensor has a selective binding affinity for SUMO compared toubiquitin proteins or other ubiquitin-like proteins.

The chimeric polypeptide sensors of the present invention can bedesigned to selectively bind to one or a subset of a group of ubiquitinproteins and ubiquitin-like proteins. The selective qualities of thechimeric polypeptides can depend on the sequences that bind to ubiquitinor ubiquitin-like proteins of which they are comprised, as well as thelinkers that connect the sequences. For example, a chimeric polypeptidesensor can be designed to selectively bind to free ubiquitin proteincompared to conjugated ubiquitin protein, and compared to ubiquitin-likeproteins. In some embodiments, this can be achieved by linking three,non-overlapping ubiquitin binding domains with two linkers (see Examples1 and 2). The tIVR, tIDR, and tISR chimeric polypeptide sensors areexamples of chimeric polypeptide sensors that selectivity bind to freeubiquitin as compared to conjugated ubiquitin or ubiquitin-likeproteins. Both of these chimeric polypeptide sensors bind to three,non-overlapping regions of ubiquitin protein. However, the tIVR chimericpolypeptide sensor can be tailored to have a greater affinity forconjugated ubiquitin by increasing the length of a linker region (seeExample 2, FIGS. 10-12). Thus, both the sequences that bind to theubiquitin or ubiquitin-like protein and the linker length determine thebinding characteristics of the chimeric polypeptide sensor. The chimericpolypeptide receptor tSR is an example of a chimeric polypeptidereceptor that can bind to a subset of ubiquitin proteins orubiquitin-like proteins. The tSR comprises two sequences that bind toubiquitin protein and one linker. The tSR is a shorted tISR, bothchimeric polypeptide sensors comprise the S5a^(UIM) and Ruz domains, buttSR lacks the IsoT^(Znf) domain. The tSR shows a similar bindingaffinity to free and conjugated ubiquitin protein (see example 8). Yetwhile tSR does not seem to distinguish between free and conjugatedubiquitin protein, tSR does show a greater affinity for ubiquitinprotein than for non-ubiquitin proteins. This demonstrates that thenumber of sequences that bind to a ubiquitin or ubiquitin-like proteinincluded can influence the properties of a binding protein. Anotherexample of a chimeric polypeptide sensor that recognizes a subset ofubiquitin proteins or ubiquitin-like proteins is tIV L1 Cys. tIV L1 Cysshares the IsoT^(Znf) domain and Vps27^(UIM) domain of tIVR, but lacksthe Ruz binding domain. The tIV L1 Cys chimeric polypeptide selectivelybinds to free ubiquitin and free Nedd8 compared to conjugated ubiquitinprotein and conjugated ubiquitin-like protein (see FIG. 7, example 2).Thus, different chimeric polypeptide sensors can be constructed toselectively bind to one or a subset of a group of ubiquitin proteins andubiquitin like proteins.

In certain embodiments, chimeric polypeptides of the present inventionare tagged with a detectable marker, including any of those describedherein or known in the art. In particular embodiments, the detectablemarker is a fluorophore or a quencher. In particular embodiments, thedetectable marker is linked to a cysteine residue in the chimericpolypeptide.

Chimeric polypeptide sensors and competitor proteins of the presentinvention may be produced recombinantly using conventional molecular andcellular biology techniques known and available in the art. Accordingly,the present invention includes recombinant chimeric polypeptide sensorsand chimeric competitor proteins, including any of those describedherein.

Methods for Determining Amounts of Ubiquitin Protein and Ubiquitin-LikeProtein

The present invention provides chimeric polypeptide sensors that can beused in a variety of assays, including assays for determining thepresence of, an amount of, or a concentration of a ubiquitin protein ora ubiquitin-like protein in a sample. In some embodiments, the assaysdetect total ubiquitin protein, free ubiquitin protein, or conjugatedubiquitin protein, or two or more of these. In some embodiments, theassays detect total ubiquitin-like protein, free ubiquitin-like protein,or conjugated ubiquitin-like protein, or two or more of these. Thepresent invention also provides chimeric polypeptide sensors that can beused to detect increases or decreases in the amounts of a ubiquitinprotein or ubiquitin-like protein, e.g., a free or conjugated ubiquitinprotein or ubiquitin-like protein. The present invention also provideschimeric polypeptide sensors that can be used in deubiquitinase assaysand other assays to measure a deconjugation or release of a ubiquitinprotein or a ubiquitin-like protein from a ubiquitin conjugatesubstrate. The present invention also provides chimeric polypeptidesthat can be used to screen for agents that increase or decrease amountsof either free or conjugated ubiquitin protein or free or conjugatedubiquitin-like protein.

In some embodiments, the invention comprises methods to quantify aubiquitin protein or ubiquitin-like protein in vitro. In someembodiments, the total amount of the ubiquitin or the ubiquitin-likeprotein is quantified, whereas in other embodiments, the free and/orconjugated ubiquitin protein or ubiquitin-like protein is quantified.

In various embodiments of methods of the present invention, total targetubiquitin protein or total target ubiquitin-like protein is assayedusing a chimeric polypeptide sensor that binds to both the free andconjugated target ubiquitin protein or both the free and conjugatedtarget ubiquitin-like protein. In various embodiments of methods of thepresent invention, free target ubiquitin protein or free targetubiquitin-like protein is assayed using a chimeric polypeptide sensorthat preferentially binds to the free target ubiquitin protein or thefree target ubiquitin-like protein. One of skill in the art willappreciate that amounts of conjugated target ubiquitin protein orconjugated target ubiquitin-like protein may be readily determined basedon a determination of the amount of free and total ubiquitin protein orubiquitin-like protein, i.e., conjugated protein=total protein−freeprotein. Accordingly, certain assays described herein includedetermining both the amount of free and the total amount of a ubiquitinprotein or a ubiquitin-like protein. As used herein, a “target” proteinrefers to the ubiquitin protein or ubiquitin-like protein being assayed.In particular instances, one portion of a sample is assayed using achimeric polypeptide sensor that binds a both free and conjugatedubiquitin protein or a both free and conjugated ubiquitin-like proteinto determine a total amount or concentration of the ubiquitin orubiquitin-like protein in the sample, and another portion of the sampleis assayed using a chimeric polypeptide sensor that preferentially bindsto a free ubiquitin protein or a free ubiquitin-like protein todetermine an amount of the free ubiquitin protein or free ubiquitin-likeprotein in the sample. The concentration of free/total ubiquitin proteinor free/total ubiquitin-like protein can be determined based on theresults of these two assays. In addition, the amount or concentration ofconjugated ubiquitin protein or ubiquitin-like protein can also beextrapolated.

In various embodiments, methods of the present invention are used todetermine an amount of a ubiquitin protein or a ubiquitin-like proteinpresent in a sample. In particular embodiments, the sample is abiological sample. Biological samples include samples obtained from anyliving cell, tissue, organ or organism, including but not limited to,blood, serum, cells, tissues, a tissue biopsy, lung effluent, urine, orcell lysate. In certain embodiments, a cell lysate is obtained fromcultured cells, e.g., cultured eukaryotic cells.

In various embodiments, methods or assays of the present invention areperformed by measuring the direct binding of a chimeric polypeptidesensor to a ubiquitin protein or ubiquitin-like protein. In otherembodiments, methods or assays of the present invention are performed byindirectly determining the binding of a chimeric polypeptide sensor to aubiquitin protein or ubiquitin-like protein. In some embodiments, thepresent invention comprises a measurement of the displacement of acompetitor ubiquitin protein or a competitor ubiquitin-like protein fromthe chimeric polypeptide sensor. As used herein, a “competitor”ubiquitin protein or ubiquitin-like protein refers to a ubiquitinprotein or ubiquitin-like protein that is exogenous to the sample beingassayed, i.e., it is not present in the sample being assayed. In certainembodiments, the competitor ubiquitin protein or ubiquitin-like proteincompetes with the ubiquitin protein or ubiquitin-like protein present inthe same for binding to the chimeric polypeptide.

In various embodiments, a competitor ubiquitin protein or competitorubiquitin-like protein is chosen from any ubiquitin protein orubiquitin-like protein that can bind to a chimeric polypeptide sensoremployed in a competition assay, regardless of the identity of thetarget ubiquitin protein or ubiquitin-like protein. In some embodimentsthe competitor ubiquitin protein or ubiquitin-like protein is taggedwith a detectable label. In some embodiments, the competitor ubiquitinprotein or competitor ubiquitin-like protein is the same protein as thetarget ubiquitin protein or target ubiquitin-like protein to bedetected. For example, in Example 1 (see, FIG. 8) concentrations of freeubiquitin protein (the target ubiquitin protein) are measured in acompetition assay using a free ubiquitin protein tagged with afluorophore (competitor protein). In some embodiments, the targetubiquitin or ubiquitin like proteins are different. For example, inExample 2 (see FIG. 9) the target ubiquitin protein or targetubiquitin-like protein included free ubiquitin protein, conjugatedubiquitin protein, and Nedd8, but all three were measured in thepresence of a free ubiquitin-tagged by a fluorophore. Conjugatedubiquitin and Nedd 8 (the targets) could be detected by the chimericpolypeptide sensor when the competitor was a free ubiquitin protein.These examples illustrate that the specific competitor protein does notneed to be identical to a target protein, so long as both the target andthe competitor can bind to the same sensor, and so long as the sensorcan bind either the target or the competitor at a given time, but notboth simultaneously.

In some embodiments, the present invention provides method for thedetection of total ubiquitin proteins, free ubiquitin proteins,conjugated ubiquitin proteins, total ubiquitin-like proteins, freeubiquitin-like proteins, or conjugated ubiquitin-like proteins throughthe detection of a detectable label. In some embodiments, the chimericpolypeptide is tagged with a detectable label. In some embodiments, thedetectable label is a fluorophore. In some embodiments, a competitorubiquitin protein or a competitor ubiquitin-like protein is tagged witha detectable label, e.g., a fluorophore. In some embodiments, thedetection comprises measuring fluorescence intensity. In someembodiments, the detection comprises measuring fluorescence anisotropy.In particular embodiments, both the chimeric polypeptide sensor and acompetitor ubiquitin protein or a competitor ubiquitin-like protein arelabeled with a detectable label. For example, one may be labeled with afluorescence emitter and the other labeled with a fluorescence quencher,e.g., as used in FRET. In certain embodiments, the detection occurs at asingle point in time, e.g., upon completion of the assay. In otherembodiments, the detection occurs at two or more time points, or at twoor more regular time intervals, during the assay.

The use of detectable labels is well known in the art. Detectable labelsmay be used according to the invention. Methods for conjugatingpolypeptides and detectable labels are well known in the art, as aremethods for imaging using detectable labels. Chimeric polypeptidesensors tagged with a detectable label may be employed in a wide varietyof assays, employing a wide variety of labels. In some embodiments ofthe present invention, detection of a species of ubiquitin protein orubiquitin like protein can facilitated by attaching a detectable labelto the chimeric polypeptide sensor. In some embodiments, detection of aspecies of ubiquitin protein or species of ubiquitin like protein can befacilitated by attaching a detectable label to a competitor ubiquitinprotein or a competitor ubiquitin-like protein.

Examples of detectable labels include but are not limited toradionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, 0-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin,coumarin, Alexa488, Oregon green 488, rhodamine green, Alexa 532, Cy3,Bodipy 588/586, Alexa586, TAMRA, Rox, Alexa 594, Texas red, Bodipy630/650, Cy5, Alexa647, IR Dye 680, IR Dye 680, IR Dye 700 DX, Cy5.5,Alexa 750, IR Dye 800CW, IR Dye 800, Atto 532, Atto 465; an example of aluminescent material is luminol; examples of bioluminescent materialsinclude luciferase, luciferin, and aequorin; and examples of suitableradioactive material include 125 I, 131 I, 35 S, or 3H. In someembodiments, the detectable labels include fluorescent proteins.Suitable fluorescent proteins include TagBFP, mTagBFP2, Azurite, EBFP2,mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A,mTurquoise, mTurquoise2, monomeric Midoriishi-cyan, TagCFP, mTFPI, GFP,EGFP, Emeral, Superfolder GFP, monomeric Azami Green, TagGFP2, nUKG,nWasabi, Clover, mNeonGreen, EYFP, YFP, Citrine, Venus, SYFP2, TagYFP,monomeric Kusabira Orange, MKOK, rnKO2, mOrange, mOrange2, mRaspberry,mCherry, mStrawberry, rTangerine, tdTomato, TagRFP, TagRFP1, mApple,mRuby, m-Ruby2, TagRFP675, IFP1.4, iFRP, mKeima Red, LSS-nKate1,LSS-mKate2, mBeRFP, PA-GFP, PAmCherry1, PATagRFP, Kaede green, Kaedered, KikGR1 green, KikGR1 red, PS-CFP2, mEos2 green, mEos2 red, mEos3.2green, mEos3.2 red, PSmOrange. In some embodiments of the presentinvention, detectable labels also include quenchers suitable forfluorescence resonance energy transfer (FRET) pairings. Examples ofsuitable quenchers include Dabcyl, BHQ1, BHQ2, BHQ3, CY5Q, CY7Q,Iowablack FQ, Iowablack RQ, IR Dye QC-1, QSY35, QSKY7, QXL570, QXL610,QXL680.

In certain embodiments, the amount of a ubiquitin protein or aubiquitin-like protein in a sample is determined indirectly bycontacting a polypeptide sensor with a sample in the presence of adetectably-labeled competitor ubiquitin protein or ubiquitin-likeprotein, and the amount of the ubiquitin protein or the ubiquitin-likeprotein in the sample is assayed by measuring the displacement of thecompetitor protein from the polypeptide sensor. Thus, in someembodiments, the present invention includes assays to measure adetectable label tagged to a competitor ubiquitin protein or acompetitor ubiquitin-like protein, wherein a property of the detectablelabel changes when the competitor ubiquitin protein or the competitorubiquitin-like protein is bound to a chimeric polypeptide sensor ascompared to when the ubiquitin protein or ubiquitin-like protein is notbound to the chimeric polypeptide. Examples of such competitor ubiquitinproteins are described herein and include, e.g., fluorescein-taggedubiquitin (S20C), Atto532-tagged ubiquitin (S20C), and Alexa488-taggedubiquitin (S20C).

In certain embodiments, the amount of a ubiquitin protein or aubiquitin-like protein in a sample is determined either directly bycontacting a detectably-labeled polypeptide sensor with a sample, andthe amount of the ubiquitin protein or the ubiquitin-like protein in thesample is assayed by measuring a change in the detectable label. Thus,in some embodiments, the present invention includes assays to measure adetectable label tagged to a chimeric polypeptide sensor, wherein aproperty of the detectable label changes when the chimeric polypeptidesensor is bound to a ubiquitin protein or a ubiquitin-like protein ascompared to when the chimeric polypeptide sensor is not bound to theubiquitin protein or the ubiquitin-like protein. Examples of suchchimeric polypeptide sensors are described herein and include, e.g.,Atto532-tagged tIVR L1 Cys, Atto532-tagged tIV L1 Cys, Alexa488-taggedtIVR (R218C), and Atto532-tISR L1 Cys.

In some embodiments, the present invention comprises methods ofdetecting a ubiquitin protein or ubiquitin-like protein (e.g., total orfree), using a chimeric polypeptide sensor in assays that measurefluorescence intensity. In some embodiments, the chimeric polypeptidesensor binds a fluorophore-tagged competitor ubiquitin protein or afluorophore-tagged competitor ubiquitin-like protein, wherein thefluorescence intensity from the fluorophore-tagged competitor ubiquitinprotein or fluorophore-tagged competitor ubiquitin-like protein isdifferent when the fluorophore-tagged competitor ubiquitin orfluorophore-tagged competitor ubiquitin-like protein is bound to thechimeric polypeptide as compared to when the fluorophore-taggedcompetitor ubiquitin protein or fluorophore-tagged competitorubiquitin-like protein is not bound to the chimeric polypeptide. In someembodiments, the binding of the chimeric polypeptide decreases thefluorescence intensity of the fluorophore-tagged competitor ubiquitinprotein or fluorophore-tagged competitor ubiquitin-like protein. In someembodiments, the binding to the chimeric polypeptide increases thefluorescence intensity of the fluorophore-tagged competitor ubiquitinprotein or fluorophore-tagged competitor ubiquitin-like protein.

In other related embodiments, the chimeric polypeptide sensors arefluorophore-tagged, and the fluorescence intensity from thefluorophore-tagged chimeric polypeptide sensor is different when boundto a ubiquitin protein or ubiquitin-like protein as compared to when itis not bound to the ubiquitin protein or ubiquitin-like protein. In someembodiments, the binding of the chimeric polypeptide decreases itsfluorescence intensity. In some embodiments, the binding of the chimericpolypeptide increases its fluorescence intensity.

Fluorescent probes are widely used in the art and are incorporated intoa wide array of techniques to study biological and biochemicalprocesses. Fluorescence intensity has been widely applied over the lasttwo decades due to the vast development of new fluorophores. Typically,an optical system illuminates and excites the sample at a specificwavelength selected by a high performance optical filter. As a result,the sample emits light and a second optical system collects the emittedlight. Usually, the emitted light is of lower energy and thus iscomposed of a longer wavelength than the excitation light. Commercialinstruments to measure fluorescence intensity are widely available, andmany different products are available to measure fluorescence intensityin different experimental settings. Fluorescence may be measured by anymeans known or available in the art, including but not limited tofluorescence intensity, fluorescence anisotropy, and FRET.

Fluorescence polarization or anisotropy is a highly sensitive method forthe detection of a species of ubiquitin protein or a species ofubiquitin-like protein according to the present invention. Whenfluorescent molecules are excited with plane polarized light, they emita majority of light in the same polarized plane, provided that themolecule remains stationary during the lifetime of the excited state (4nanoseconds in the case of fluorescein). However, if the moleculerotates or tumbles out of the plane of the exciting polarized lightduring the excited state, light is emitted in a different plane fromthat of the initial excitation. The degree to which the fluorescenceemission vector moves from, e.g., a vertical to a horizontal plane isdirectly related to the mobility of the fluorescently labeled molecule.That is, if the fluorescently labeled molecules are large, they movevery little and the emitted light remains highly polarized with respectto the excitation plane. By contrast, if the fluorescently labeledmolecules are small, they rotate or tumble faster, and the resultingemitted light is depolarized relative to the excitation plane. Becauseanisotropy is a general property of fluorescent molecules, an advantageof measuring fluorescent anisotropy is that readouts are less dyedependent and less susceptible to environmental interferences such as pHchanges than assays based on fluorescence intensity measurements. Insome embodiments of the invention, a fluorophore tagged to a chimericpolypeptide sensor or a ubiquitin protein will have a differentanisotropy when the chimeric polypeptide sensor is bound to ubiquitinprotein as opposed to unbound, and anisotropy can be used as ameasurement for the assays of the present invention. Measurement offluorescence anisotropy can easily be performed by persons skilled inthe art. Furthermore, measuring fluorescence anisotropy is particularlysuitable for high-throughput applications. For example, commercialinstruments exist that can measure polarization of samples present in96-well microtiter dishes.

FRET is a distance-dependent interaction between the electronic excitedstates of two dye molecules in which excitation is transferred from adonor molecule to an acceptor molecule without emission of a photon. Theefficiency of FRET is dependent on the inverse sixth power of theintermolecular separation, making it useful over distances comparable tothe dimensions of biological macromolecules. Thus, FRET is an importanttechnique for investigating a variety of biological phenomena thatproduce changes in molecular proximity. Some embodiments of theinvention comprise the binding of a ubiquitin protein tagged with afirst detectable label to a chimeric polypeptide sensor bound with asecond detectable label. In some embodiments, one of the labels is adonor dye (e.g., fluorescence donor) and one of the labels is anacceptor dye (e.g., fluorescence quencher). The absorbance spectrum ofan acceptor dye overlaps with the emission spectrum of a proximalintramolecular or intermolecular donor dye such that the fluorescence ofthe donor dye is substantially diminished, or quenched. Acceptor dyesmay or may not be fluorescent themselves. Fluorescent acceptor dyesallow for ratio analysis of fluorescence from the donor and acceptordyes by two-channel detection. In some applications, non-fluorescentacceptor dyes may be advantageous, because they eliminate backgroundfluorescence that results from direct acceptor excitation.

In some embodiments, the present invention includes methods of detectingubiquitin proteins or ubiquitin-like proteins with chimeric polypeptidesensors in assays that measure fluorescence anisotropy. In someembodiments, the chimeric polypeptide sensors bind a fluorophore-taggedubiquitin protein or a fluorophore-tagged ubiquitin-like protein (e.g.,a competitor protein), wherein the fluorescence anisotropy measured fromthe fluorophore-tagged ubiquitin protein or fluorophore-taggedubiquitin-like protein is different when the fluorophore-taggedfluorophore-tagged ubiquitin or ubiquitin-like protein is bound to thechimeric polypeptide as compared to when the fluorophore-taggedubiquitin protein or fluorophore-tagged ubiquitin-like protein is notbound to the chimeric polypeptide. In some embodiments, binding to thechimeric polypeptide sensor decreases the fluorescence anisotropymeasured from the fluorophore-tagged ubiquitin protein orfluorophore-tagged ubiquitin-like protein. In some embodiments, bindingto the chimeric polypeptide increases the fluorescence anisotropymeasured from the fluorophore-tagged ubiquitin protein or thefluorophore-tagged ubiquitin-like protein. In some embodiments, thedissociation of the binding of the chimeric polypeptide decreases thefluorescence anisotropy measured from the fluorophore-tagged ubiquitinprotein or fluorophore-tagged ubiquitin-like protein. In someembodiments, the dissociation of the binding to the chimeric polypeptideincreases the fluorescence anisotropy measured from thefluorophore-tagged ubiquitin protein or the fluorophore-taggedubiquitin-like protein. In other related embodiments, the chimericpolypeptide sensors are fluorophore-tagged, and the fluorescenceanisotropy from the fluorophore-tagged chimeric polypeptide sensor isdifferent when bound to a ubiquitin protein or ubiquitin-like protein ascompared to when it is not bound to the ubiquitin protein orubiquitin-like protein. In some embodiments, the binding of the chimericpolypeptide decreases its fluorescence anistropy. In some embodiments,the binding of the chimeric polypeptide increases its fluorescenceanisotropy.

Certain embodiments of assays of the present invention utilizecompetition assays. In some embodiments, a premise of the competitionassay is that the binding of the ubiquitin protein being detected to thechimeric polypeptide sensor excludes the binding of a competitorubiquitin protein tagged with a detectable label to the chimericpolypeptide sensor. When the species of ubiquitin protein to be detectedis not present, there is a high degree of binding between the chimericpolypeptide sensor and the detectable label-tagged competitor ubiquitinprotein. When concentrations of the ubiquitin protein being detected arepresent, they compete with the detectable-tagged competitor ubiquitinproteins for binding to the chimeric polypeptide sensor. Thus, thepresence of the ubiquitin protein being detected in the sample beingassayed results in a lower degree of binding between the chimericpolypeptide and the tagged competitor ubiquitin protein. Since theproperties of the detectable label attached to the competitor ubiquitinprotein change when the tagged competitor ubiquitin protein is bound tothe chimeric polypeptide sensor, the amount of tagged competitorubiquitin protein bound to the chimeric sensor can be determined bymeasuring the properties of the detectable label. From this measurement,the amount of the ubiquitin protein being detected can also bedetermined. In some embodiments, the detectable label is a fluorophore,where in the amount of the ubiquitin protein being detected can bedetermined by measuring a property of fluorescence emitted by thefluorophore. In some embodiments, the measurement quantifies fluorescentintensity. In some embodiments, the measurement quantifies fluorescentanisotropy. In some embodiments, the chimeric polypeptide is tagged witha detectable label that can be paired with the fluorophore-taggedcompetitor ubiquitin for FRET. In some embodiments, the measurement ofthe detectable label can be compared to a designated control todetermine a relative amount of the ubiquitin proteins being detected. Insome embodiments, the measurement of the detectable label can becompared to values determined from a set of standards with knownquantities of the ubiquitin protein being detected to determine theabsolute amount of the ubiquitin protein being detected in the sample.

According to various embodiments of the invention, any chimericpolypeptide sensor of the invention can be incorporated into acompetition assay as described above. This includes, but it not limitedto chimeric polypeptide sensors comprising two or more sequences thatbind a target ubiquitin protein; comprising two or more sequences thatbind a target ubiquitin-like protein; comprising a sequence that bindsubiquitin C-terminal; comprising a sequence that binds the ubiquitinhydrophobic patch; comprising a sequence that binds to surface nearubiquitin Asp58; comprising a sequence that binds the ubiquitinC-terminal and a sequence that binds the ubiquitin hydrophobic patch;comprising a sequence that binds to the ubiquitin C-terminal and asequence that binds to the surface near ubiquitin Asp58; comprising asequence that binds to the ubiquitin hydrophobic patch and a sequencethat binds to the surface near ubiquitin Asp58; comprising a sequencethat binds the ubiquitin C terminal, a sequence that binds the ubiquitinhydrophobic patch, and a sequence that binds to the surface nearubiquitin Asp58; comprising the ZnF UBP binding domain from IsopeptidaseT, comprising the Ruz domain from Rabex-5; comprising the ubiquitininteracting motif from Vps27; comprising the Ruz domain from Rabex-5 andthe ubiquitin interacting motif from Vps27; comprising the ZnF UBPdomain from Isopeptidase T and the Ruz domain from Rabex5; comprisingthe ubiquitin interacting motif from Vps27 and the Ruz domain fromRabex5; comprising the ZnF UBP binding domain from Isopeptidase T, theRuz domain from Rabex-5, and the ubiquitin interacting motif from Vps27;comprising the ubiquitin binding associated domain from Dsk2; comprisingthe ZnF UBP binding domain from Isopeptidase T and the ubiquitinassociated domain from Dsk2; comprising the Ruz domain from Rabex-5 andthe ubiquitin associated domain from Dsk2; comprising the ZnF UBPbinding domain from Isopeptidase T, the Ruz domain from Rabex-5, and theubiquitin associated domain from Dsk2; comprising the ubiquitininteracting motif from S5a; or comprising the ZnF UBP binding domainfrom Isopeptidase T, the Ruz domain from Rabex-5, the ubiquitininteracting motif from S5a. Furthermore, this includes, but it notlimited to: tIVR, tIDR, tISR, tSR, and tIV. In some embodiments, thechimeric polypeptide comprises a detectable label. In some embodiments,the competition assays also utilize a ubiquitin protein orubiquitin-like protein tagged with a detectable label (e.g., acompetitor protein). In some embodiments, the include competition assaysutilize a ubiquitin or ubiquitin-like protein tagged with a fluorophore.In some embodiments, the competition assays utilize a chimericpolypeptide sensor with a detectable label and a ubiquitin protein or aubiquitin-like protein tagged with a detectable label. In someembodiments, these include competition assays utilizing FRET imagingtechniques.

In some embodiments, the present invention comprises methods ofdetecting ubiquitin protein or ubiquitin-like protein by detectingchimeric polypeptide sensor tagged with a detectable label. In someembodiments, the detection method comprises a direct titration assay.Direct titration assays are well known in the art, and any such methodto perform titration assay is suitable. In some embodiments, thetitration assay measures a ubiquitin protein to be determined. In someembodiments, the titration assay measures a ubiquitin-like protein to bedetermined. In some embodiments of the present invention, competitiveassays comprise measuring fluorescence intensity. In some embodiments ofthe present invention, competitive assays comprise measuringfluorescence anisotropy.

In some embodiments of the present invention, a premise of the titrationassay is that the binding of a ubiquitin protein or ubiquitin-likeprotein by the detectable label-tagged chimeric polypeptide alters theproperties of the detectable label. For example, when the ubiquitinprotein to be detected is not present, there is no degree of bindingbetween the chimeric polypeptide sensor and the ubiquitin protein. Whenamounts of the ubiquitin protein being detected are present, they bindto the chimeric polypeptide sensor with a detectable label. Since thebinding of the ubiquitin protein with the chimeric polypeptide sensortagged with a detectable label changes the properties of detectablelabel, the amount of the ubiquitin protein bound to the chimeric sensorcan be determined by measuring the detectable label. From thismeasurement, the amount of the ubiquitin protein in the sample can alsobe determined. In some embodiments, the detectable label is afluorophore, wherein the amount of the ubiquitin protein can bedetermined by measuring a property of fluorescence emitted by thefluorophore. In some embodiments, the measurement quantifies fluorescentintensity. In some embodiments, the measurement quantifies fluorescentanisotropy. In some embodiments, the measurement of the detectable labelcan be compared to a designated control to determine a relative amountof the ubiquitin protein being detected. In some embodiments, themeasurement of the detectable label can be compared to values determinedfrom a set of standards with known quantities of the ubiquitin proteinbeing detected to determine the absolute amount of the ubiquitin proteinbeing detected in the sample.

According to some embodiments of the invention, any chimeric polypeptidesensor of the invention can be used in a direct titration assay asdescribed above. This includes, but it not limited to chimericpolypeptide sensors comprising two or more sequences that bind ubiquitinprotein; comprising two or more sequences that bind ubiquitin-likeprotein; comprising a sequence that binds ubiquitin C-terminal;comprising a sequence that binds the ubiquitin hydrophobic patch;comprising a sequence that binds to surface near ubiquitin Asp58;comprising a sequence that binds the ubiquitin C-terminal and a sequencethat binds the ubiquitin hydrophobic patch; comprising a sequence thatbinds to the ubiquitin C-terminal and a sequence that binds to thesurface near ubiquitin Asp58; comprising a sequence that binds to theubiquitin hydrophobic patch and a sequence that binds to the surfacenear ubiquitin Asp58; comprising a sequence that binds the ubiquitin Cterminal, a sequence that binds the ubiquitin hydrophobic patch, and asequence that binds to the surface near ubiquitin Asp58; comprising theZnF UBP binding domain from Isopeptidase T, comprising the Ruz domainfrom Rabex-5; comprising the ubiquitin interacting motif from Vps27;comprising the Ruz domain from Rabex-5 and the ubiquitin interactingmotif from Vps27; comprising the ZnF UBP domain from Isopeptidase T andthe Ruz domain from Rabex5; comprising the ubiquitin interacting motiffrom Vps27 and the Ruz domain from Rabex5; comprising the ZnF UBPbinding domain from Isopeptidase T, the Ruz domain from Rabex-5, and theubiquitin interacting motif from Vps27; comprising the ubiquitin bindingassociated domain from Dsk2; comprising the ZnF UBP binding domain fromIsopeptidase T and the ubiquitin associated domain from Dsk2; comprisingthe Ruz domain from Rabex-5 and the ubiquitin associated domain fromDsk2; comprising the ZnF UBP binding domain from Isopeptidase T, the Ruzdomain from Rabex-5, and the ubiquitin associated domain from Dsk2; orcomprising the ubiquitin interacting motif from S5a; comprising the ZnFUBP binding domain from Isopeptidase T, the Ruz domain from Rabex-5, theubiquitin interacting motif from S5a. Furthermore, this includes, but itnot limited to: tIVR, tIDR, tISR, tSR, and tIV. In some embodiments, thechimeric sensor polypeptide is detectably labeled. In some embodiments,these include direct titration assays that use a chimeric polypeptidesensor tagged with a detectable label. In some embodiments, theseinclude direct titration assays use a chimeric polypeptide sensor taggedwith a fluorophore.

In some embodiments of the invention, competition assays and directtitration assays are suitable for determining an amount of a targetubiquitin protein or ubiquitin-like protein in vitro. In someembodiments, the assays described herein are suitable for determining anamount of a target ubiquitin protein or ubiquitin-like protein in asample, e.g., a biological sample. In some embodiments of the invention,the competition assays and the direct titrations assays are suitable fordetermining an amount of a target ubiquitin protein or ubiquitin-likeprotein in extracts. In some embodiments of the invention, the extractsare from cultured cells. In some embodiments of the invention, theextracts are from tissue. The preparation of extracts from cell cultureand from tissue for use in experiments as described herein are wellknown to those of skill in the art.

In certain embodiments, the present invention includes a method ofdetermining the presence of, an amount of, or a concentration of aubiquitin protein or a ubiquitin-like protein (i.e., target protein) ina sample, comprising: contacting the sample with a chimeric polypeptidesensor described herein for a period of time and directly or indirectlydetecting an amount of the ubiquitin protein or ubiquitin-like proteinbound to the chimeric polypeptide sensor.

In some embodiments, the ubiquitin protein or ubiquitin-like proteinbeing assayed is the free ubiquitin protein or ubiquitin-like protein,and the chimeric polypeptide sensor preferentially binds to the freeubiquitin protein or ubiquitin-like protein.

In some embodiments, the ubiquitin protein or ubiquitin-like proteinbeing assayed is the total ubiquitin protein or ubiquitin-like protein,including both the free and conjugated ubiquitin protein orubiquitin-like protein, and the chimeric polypeptide sensor binds toboth the free ubiquitin protein or free ubiquitin-like protein and theconjugated ubiquitin protein or conjugated ubiquitin-like protein.

In certain embodiments, the chimeric polypeptide sensor is detectablylabeled, e.g., with a fluorophore, wherein the signal emitted by thelabel is altered upon binding to the target protein, and the amount oftarget protein bound to the chimeric polypeptide sensor is measured bydetecting a change in the signal emitted by the label after contact withthe sample or throughout the period of time, e.g., when the signal isdetected at regular intervals throughout the period of time.

In other embodiments, the method comprises contacting the sample and thechimeric polypeptide sensor with a detectably labeled competitorubiquitin protein or competitor ubiquitin-like protein for a period oftime, wherein the signal emitted by the label is altered upon binding tothe chimeric polypeptide sensor, and the amount of target protein boundto the chimeric polypeptide sensor is indirectly measured by detecting achange in the signal emitted by the label after contact between thelabeled competitor protein and the chimeric polypeptide sensor in thepresence of the sample. In certain embodiments, the competitor proteinis pre-incubated with the chimeric polypeptide sensor before both arecontacted by the sample, and a change in the signal emitted reflectscompetition by the target protein in the sample for binding to thechimeric polypeptide sensor.

In other embodiments, the method comprises contacting the sample and adetectably labeled chimeric polypeptide sensor with a detectably labeledcompetitor ubiquitin protein or competitor ubiquitin-like protein for aperiod of time, wherein the signal emitted by the labels is altered uponbinding of the competitor protein to the chimeric polypeptide sensor,and the amount of target protein bound to the chimeric polypeptidesensor is indirectly measured by detecting a change in the signalemitted by the labels after contact between the labeled competitorprotein and the labeled chimeric polypeptide sensor in the presence ofthe sample. In certain embodiments, either the chimeric polypeptidesensor or the competitor protein is labeled with a fluorescence emitter,and the other of the two is labeled with a quencher, wherein the pair oflabels may be used in FRET. In certain embodiments, the competitorprotein is pre-incubated with the chimeric polypeptide sensor beforeboth are contacted by the sample, and a change in the signal emittedreflects competition by the target protein in the sample for binding tothe chimeric polypeptide sensor.

In particular embodiments of any of the methods described herein, thesample is incubated with the chimeric polypeptide sensor and,optionally, the competitor protein, under conditions and for a durationof time sufficient to allow binding of the chimeric polypeptide sensorto the target ubiquitin protein or target ubiquitin-like protein in thesame and/or the competitor protein. In certain embodiments, theconditions are any of those described in the accompanying Examples. Incertain embodiments, the duration of time is at least 1 minute, at least10 minutes, at least 30 minutes, at least one hour, at least two hours,or at least four hours. In certain embodiments, the assays are conductedin a solution approximating or having physiological conditions,including use of phosphate-buffered saline (25 mM Na phosphate pH 7.4,150 mM NaCl) at a pH of 7.4, or Na HEPES or Na phosphate buffer at a pHof about 7.0, about 7.4, or about 7.5. In particular embodiments, assaymixtures additionally contain 0.1 mM tris-(carboxyethyl)phosphinehydrochloride to protect proteins from oxidation and 0.05% (w/v) Brij35plus 0.2 mg/ml ovalbumin to reduce non-specific adsorption of proteinsto surfaces (e.g., to quartz cuvette walls or plastic pipet tips). Inparticular embodiments, the binding step of the assays is performed atabout 25 degrees C. or, for real-time deubiquitinase assays, at about 25degrees C., or at about 30 degrees C., or at about 37 degrees C.

In particular embodiments of any of the methods described herein, theamount of target ubiquitin protein or target ubiquitin-like protein inthe sample is determined by comparing the amount of detected signal to apredetermined value or set of values, or to a control value. In someembodiments, the predetermined control values are determined byperforming the same assay using various known amounts of target proteininstead of a test sample, to generate a set of known values. In someembodiments, a control value is determined by performing an assayconcurrent with the test assay, but using a negative control instead ofa test sample. In certain embodiments, a negative control is a samplethat does not include the target ubiquitin protein or ubiquitin-likeprotein. In certain embodiments, a negative control is a protein that isnot bound by the chimeric polypeptide sensor.

In particular embodiments of any of the methods described herein, theprotein being detected is human ubiquitin protein, and the chimericpolypeptide sensor is a tIVR, tIVR L1 Cys, tIVR (R218C), tIDR, tISR, ortSR chimeric polypeptide sensor.

In particular embodiments of any of the methods described herein, theprotein being detected is Nedd8, and the chimeric polypeptide is a tIVchimeric polypeptide sensor.

In some embodiments, the present invention includes a method todetermine the presence of deubiquitinase activity or an amount ofdeubiquitinase activity. In some embodiments of the invention, thedeubiquitinase assay is an in vitro assay to determine thedeubiquitinase activity present in a sample or the deubiquitinaseactivity of a known, putative, or candidate deubiquitinase. In certainembodiments, the assay is used to screen a library comprising aplurality of compounds to identify a compound having deubiquitinaseassay or a compound that activates or inhibits activity of adeubiquitinase enzyme. In particular embodiments, the library comprisesa plurality of peptides or polypeptides.

In other embodiments, a deubiquitinase assay of the present inventionmay be used to assess the substrate promiscuity of a known, putative orcandidate deubiquitinase by determining its activity using a pluralityof different conjugated ubiquitin proteins and/or conjugatedubiquitin-like proteins. For example, the assay may be performed todetermine the deubiquitinase activity of the known, putative orcandidate deubiquitinase against ubiquitin proteins conjugated to aplurality of different substrate polypeptides, e.g., to determinewhether the known, putative or candidate deubiquitinase specificallytargets only one or a subset of ubiquitin-conjugated polypeptides. Incertain instances, the assay is performed in multi-well dishes, e.g.,96-well plates, wherein each of the plurality of differentubiquitin-conjugated polypeptides is present in a discrete well.

In some embodiments, a deubiquitinase is defined as a protein withprotease activity that removes ubiquitin and polyubiquitin chains fromubiquitinated proteins. Thus, provided a source of conjugated ubiquitin,a deubiquitinase will deconjugate the conjugated ubiquitin, and therebyincrease the amount of free ubiquitin. In some embodiments of theinvention, deubiquitinase activity is detected by a chimeric polypeptidesensor with preferential binding to free ubiquitin protein or freeubiquitin-like protein as compared to the conjugated ubiquitin proteinor conjugated ubiquitin-like protein. In some embodiments, thedeubiquitin assay is performed with any of the techniques or assaysdescribed, include competition assays and direct titration assays todetect free ubiquitin. In some embodiments, the deubiquitinase assayuses a chimeric polypeptide sensor tagged with a detectable label. Insome embodiments, a competitor ubiquitin protein is tagged with adetectable label. In some embodiments, the deubiquitinase assaycomprises the measurement of fluorescence properties. In someembodiments, the deubiquitinase assay comprises the measurement offluorescence anisotropy. In some embodiments, the deubiquitinase assaycomprises FRET.

A deubiquitinase assay detects deubiquitinase activity, which is theactivity that breaks the covalent bonds between ubiquitin proteins andnon-ubiquitin proteins. In a given sample, deubiquitinase activityincreases the amount of free ubiquitin in the sample. In essence, adeubiquitinase assay determines if an amount of free ubiquitin isgenerated from a source of conjugated ubiquitin by a known or candidatedeubiquitinase. Thus, any methods of the present invention that candetect free ubiquitin can be purposed towards detecting deubiquitinaseactivity. One strategy to detect deubiquitinase activity is to measurethe amount of free ubiquitin in a sample containing a known or candidatedeubiquitinase, and comparing it the amount of free ubiquitin in anegative control sample that does not contain the known or candidatedeubiquitinase, whereby a greater amount of free ubiquitin in thepresence of the known or candidate deubiquitinase compared to thenegative control would confirm that the known or candidate is adeubiquitinase. Such a strategy could be employed by methods frommultiple embodiments of the invention, including direct titration andcompetition assays. A second strategy is to measure the amount of freeubiquitin at two or more time periods in a mixture containing a known orcandidate deubiquitinase, a source of conjugated ubiquitin, and thechimeric polypeptide sensor that preferentially binds to free ubiquitinas compared to conjugated ubiquitin. In some embodiments of theinvention, two or more time points are monitored over a period of time.In some embodiments, the methods comprise taking measurements of freeubiquitin in a mixture at regular intervals over a period of time. Ifgreater amounts of free ubiquitin are measured at later time pointscompared to earlier time points, it indicates the presence ofdeubiquitinase activity. In some embodiments, the measurements of freeubiquitin are performed before the known or candidate deubiquitinasecontacts the mixture. In other embodiments, measurements are taken afterthe known or candidate deubiquitinase contacts the mixture. In someembodiments, the measurements are take both before and after the knownor candidate deubiquitinase contacts the mixture.

In some embodiments of the invention, the methods comprise a series ofsteps, in any order, comprising contacting a chimeric polypeptide sensorthat preferentially binds to free ubiquitin to a mixture, contacting asource of conjugated ubiquitin to the mixture, contacting a known orcandidate deubiquitinase to the mixture for a period of time, andmeasuring the amount of free ubiquitin. In some embodiments, the methodscomprise contacting a mixture first with a chimeric polypeptide sensortagged with a detectable label and contacting the mixture with a sourceof conjugated ubiquitin, and next contacting the mixture with the knownor candidate deubiquitinase. In other embodiments, the chimeric sensorcontacts the mixture after the known or candidate deubiquitinasecontacts the mixture. In some embodiments, the amount of ubiquitin isdetermined is determined through direct titration experiments, whereinthe chimeric polypeptide sensor is tagged with a detectable label. Insome embodiments, the amount of free ubiquitin is determined with acompetition assay, wherein a competitor ubiquitin protein orubiquitin-like protein tagged with a detectable label contacts themixture. In some embodiments, the amount of free ubiquitin is measuredwith FRET, wherein the chimeric polypeptide sensor is tagged with adetectable label and the competitor ubiquitin protein or competitorubiquitin-like protein.

In some embodiments of the invention, the term candidate deubiquitinaserefers to an enzyme suspected to possess deubiquitinase activity. Insome embodiments, candidate deubiquitinase refers to an agent beingtested for or suspected to modify the deubiquitinase activity of theknown deubiquitinase. In some embodiments, such agents can include, butare not limited to: small molecules and pharmaceutical compounds,proteasome inhibitors, RNA or DNA oligonucleotides, antibodies, or otherproteins. In such experiments, negative controls would include a groupwith no deubiquitinase, and a control with the known deubiquitinase butnot the agent. If a presence of the agent in the mixture results in adifferent amount of free ubiquitin, either a greater or lower amount offree ubiquitin, then it can be concluded that the agent modifies theactivity of the deubiquitinase. As indicated, methods of the presentinvention may also be practiced using known deubiquitinases, e.g., tomeasure their activity, e.g., in the presence of another compound, or todetermine their substrate specificity.

In one particular embodiment, a deubiquitinase assay of the presentinvention comprises bringing into contact: (i) a chimeric polypeptidesensor that preferentially binds to a free ubiquitin protein or a freeubiquitin-like protein as compared to the conjugated ubiquitin proteinor conjugated ubiquitin-like protein; (ii) one or more conjugatedubiquitin proteins or conjugated ubiquitin-like proteins; and (iii) oneor more known or candidate ubiquitinases, for a period of time, e.g., aperiod of time sufficient to allow binding of the chimeric polypeptidesensor to free ubiquitin protein or free ubiquitin-like protein anddeconjugation of ubiquitin. In particular embodiments, the chimericpolypeptide sensor is detectably labeled, and the signal generated bythe label changes when the sensor is bound by free ubiquitin. In otherembodiments, the ubiquitin protein or ubiquitin-like protein is labeled,and the signal generated by the label changes when the protein is boundby the chimeric polypeptide sensor. In other embodiments that utilize anindirect measurement, a labeled competitor free ubiquitin or competitorfree ubiquitin-like protein is also brought into contact with (i)-(iii),wherein the signal generated by the label changes when the competitorprotein is bound by the sensor as compared to when it is not bound bythe sensor.

In some embodiments of the invention, a deubiquitinase assay ispreformed using a conjugated ubiquitin protein with a detectable labeltagged on the ubiquitin protein. In some embodiments, the detectablelabel is a fluorophore. In some embodiments, the deubiquitinase assaycomprises contacting the tagged conjugated ubiquitin protein with achimeric polypeptide sensor that preferentially binds free ubiquitinprotein compared to conjugated ubiquitin protein, and contacting taggedconjugated ubiquitin protein with the tagged conjugated ubiquitinprotein, and contacting the tagged the conjugated ubiquitin with a knownor candidate deubiquitinase. In this deubiquitinase assay,deubiquitinase activity converts the tagged conjugated ubiquitin intotagged free ubiquitin. The newly free tagged ubiquitin binds to thechimeric polypeptide sensor, resulting in a detectable change in asignal from the detectable label. Increased binding of tagged freeubiquitin to the chimeric polypeptide sensors is a positive indicator ofdeubiquitinase activity in this deubiquitinase assay.

In each of these assays, the amount of free ubiquitin may be determinedby measuring the amount of detectable label. In certain embodiments, thepresence of or amount of free ubiquitin is determined by measuring thechange in the amount of label (e.g., fluorescence intensity, anisotropy,or FRET) detected at two or more different time points during the assay.In certain embodiments, the presence of or amount of free ubiquitin isdetermined by comparing the amount of label detected to a predeterminedvalue or to a control value. Generally, an increase in the amount offree ubiquitin protein or free ubiquitin-like protein detected indicatesthe presence of deubiquitinase activity and, thus, identifies a compoundas being a deubiquitinase.

According to some embodiments of the invention, any chimeric polypeptidesensor of the invention can be incorporated into a deubiquitinase assayas described above. This includes, but it not limited to chimericpolypeptide sensors: comprising two or more sequences that bindubiquitin protein; comprising a detectable label; comprising two or moresequences that bind ubiquitin-like protein; comprising a sequence thatbinds ubiquitin C-terminal; comprising a sequence that binds theubiquitin hydrophobic patch; comprising a sequence that binds to surfacenear ubiquitin Asp58; comprising a sequence that binds the ubiquitinC-terminal and a sequence that binds the ubiquitin hydrophobic patch;comprising a sequence that binds to the ubiquitin C-terminal and asequence that binds to the surface near ubiquitin Asp58; comprising asequence that binds to the ubiquitin hydrophobic patch and a sequencethat binds to the surface near ubiquitin Asp58; comprising a sequencethat binds the ubiquitin C terminal, a sequence that binds the ubiquitinhydrophobic patch, and a sequence that binds to the surface nearubiquitin Asp58; comprising the ZnF UBP binding domain from IsopeptidaseT, comprising the Ruz domain from Rabex-5; comprising the ubiquitininteracting motif from Vps27; comprising the Ruz domain from Rabex-5 andthe ubiquitin interacting motif from Vps27; comprising the ZnF UBPdomain from Isopeptidase T and the Ruz domain from Rabex5; comprisingthe ubiquitin interacting motif from Vps27 and the Ruz domain fromRabex5; comprising the ZnF UBP binding domain from Isopeptidase T, theRuz domain from Rabex-5, and the ubiquitin interacting motif from Vps27;comprising the ubiquitin binding associated domain from Dsk2; comprisingthe ZnF UBP binding domain from Isopeptidase T and the ubiquitinassociated domain from Dsk2; comprising the Ruz domain from Rabex-5 andthe ubiquitin associated domain from Dsk2; comprising the ZnF UBPbinding domain from Isopeptidase T, the Ruz domain from Rabex-5, and theubiquitin associated domain from Dsk2; comprising the ubiquitininteracting motif from S5a; comprising the ZnF UBP binding domain fromIsopeptidase T, the Ruz domain from Rabex-5, the ubiquitin interactingmotif from S5a; further, this includes, but it not limited to: tIVR,tIDR, tISR, tSR, and tIV. In some embodiments, these includedeubiquitinase assays that comprise a ubiquitin protein or ubiquitinlike protein tagged with a detectable label. In some embodiments, theseinclude deubiquitinase assays that comprise a ubiquitin orubiquitin-like protein tagged with a fluorophore. In some embodiments,these include deubiquitinase assays that comprise a chimeric polypeptidesensor with a detectable label and a ubiquitin protein or a ubiquitinlike protein tagged with a detectable label. In some embodiments, theseinclude deubiquitinase assays comprising FRET imaging techniques.

In some embodiments of the invention, competition, direct titration, anddeubiquitinase assays can be incorporated into high throughput screensto detect novel compounds or agents that influence ubiquitin proteins orubiquitin-like proteins. In some embodiments, the invention includes anyof the methods to detect free ubiquitin protein or free ubiquitin-likeprotein described herein in concert with screens to evaluate the effectsof different agents or compounds on pools, e.g., amounts, of freeubiquitin. In particular embodiments, a library of compounds or agentsis screened to identify a compound or agent that either increases ordecreases ubiquitination or deubiquitination of a substrate polypeptide.In some embodiments, the agents or compounds are small molecules, and insome embodiments, the agents or compounds are peptides or polypeptides.

Any methods of the present invention that can detect free ubiquitin orubiquitin-like protein can be purposed towards a high throughput screento detect agents that influence conjugation or deconjugation ofubiquitin or ubiquitin-like proteins. One strategy to detect modifiersof conjugation or deconjugation of ubiquitin or ubiquitin-like proteinsis to measure the amount of free ubiquitin in a sample containing acandidate agent, and compare it the amount of free ubiquitin in anegative control sample that does not contain the candidate agent,whereby a greater or lesser amount of free ubiquitin in the presence ofthe candidate agent compared to the negative control would confirm thatthe candidate influences conjugation or deconjugation. Such a strategyis employed according to certain embodiments of any of the methods ofthe invention, including direct titration and competition assays.

In some embodiments of the invention, a candidate agent is tested priorto detecting free ubiquitin. In some embodiments, agents can include,but are not limited to, small molecules and pharmaceutical compounds,DNA or RNA oligonucleotides, methods of gene silencing, proteins, orantibodies. In some embodiments, the candidate agent is contacted to anorganism or a tissue before a tissue sample from the organism or thetissue is collected. In such cases, the agent can be administered to anexperimental organism, including, but not limited to, nematoads,fruitflies, mice, rats, or plants. In some embodiments, the agent can betested in cultured cells prior to preparing the cells as an extract. Insome embodiments the agent contacts an extract prepared from a cellculture or tissue before the chimeric sensor contacts the extract. Insome of these embodiments, the agent is tested on biological processesthat occur without the presence of a chimeric polypeptide sensor and,optionally, a competitor protein. In such screens, the chimeric sensoris employed to determine the effect of the agent by detecting an amountof free ubiquitin present in extracts after biological processes oractivities the agent is suspected to influence have taken place.Chimeric polypeptide sensors tagged with a detectable label, or chimericpolypeptide sensors and competitor ubiquitin protein or competitorubiquitin-like protein tagged with a detectable label, or chimericpolypeptide sensors tagged with a detectable label and competitorubiquitin protein or ubiquitin-like protein tagged with a detectablelabel, are then contacted to the extract to determine the amount of freeubiquitin in the extract though direct titration, competition assay, orFRET methods, respectively.

According to some embodiments of the invention, one strategy todetermine if an agent influences ubiquitin protein or ubiquitin-likeprotein conjugation or deubiquitinase activity is to measure the amountof free ubiquitin or free ubiquitin-like protein in a sample comprisingan agent, and comparing it the amount of free ubiquitin in a negativecontrol sample that does not contain the agent, whereby a change in theamount of free ubiquitin in the presence of the agent as compared to thenegative control confirms that the agent alters ubiquitin protein orubiquitin-like protein conjugation or deconjugation. In this strategy,the agent is contacted to a mixture with a known entity that acts toconjugate or deconjugate ubiquitin to a substrate. For example suchentities can include, but are not limited to, proteins such asdeubiquitinases or ubiquitin E1, E2, or E3 ligases. In variousembodiments, such a strategy employs methods from various embodiments ofthe invention, including direct titration and competition assays. Asecond strategy is to measure the amount of free ubiquitin at two ormore time periods in a mixture comprising the agent, the entities thatconjugate or deconjugate ubiquitin or ubiquitin-like proteins, and achimeric polypeptide sensor. In some embodiments of the invention, twoor more time points are monitored over a period of time. In someembodiments, the methods comprise taking measurements of free ubiquitinin a mixture at regular intervals over a period of time. If the presenceof the agent reduces the change in free ubiquitin protein or freeubiquitin-like protein observed across the measurements, then the agentcan be concluded to inhibit the entity. If a larger change in the amountof free ubiquitin is observed, the agent can be concluded to enhance theconjugation or deconjugation activity of the entity.

In some embodiments of the invention, the methods comprise a series ofsteps, in any order, comprising contacting a chimeric polypeptide sensorthat preferentially binds to free ubiquitin to a mixture, contacting asource of conjugated ubiquitin to the mixture, contacting a candidateagent to the mixture comprising a known ubiquitin conjugating enzyme ordeubiquitinase for a period of time, and measuring the amount of freeubiquitin. In some embodiments, the methods comprise contacting amixture first with a chimeric polypeptide sensor tagged with adetectable label, contacting the mixture with a source of conjugatedubiquitin, and next contacting the mixture with the candidatedeubiquitinase. In other embodiments, the chimeric sensor contacts themixture after the agent contacts the mixture. In some embodiments, theamount of ubiquitin is determined through direct titration experiments,wherein the chimeric polypeptide sensor is tagged with a detectablelabel. In some embodiments, the amount of free ubiquitin is determinedwith a competition assay, wherein a competitor ubiquitin protein orubiquitin-like protein tagged with a detectable label is also contactedto the mixture. In some embodiments, the amount of free ubiquitin ismeasured with FRET, wherein the chimeric polypeptide sensor is taggedwith a detectable label and the competitor ubiquitin protein orcompetitor ubiquitin-like protein is also tagged with a detectablelabel.

Competition assays and direct titration assays utilizing a chimericpolypeptide sensor to detect free ubiquitin, including any of thosedescribed herein, can be modified into a high throughput screen to testcandidate agents for their abilities to modify pools of free ubiquitin.Such agents may include but are not limited to small molecules such as,pharmaceutical compounds, known or putative proteasome inhibitors;antibodies; proteins; nucleotides include DNA expression constructs,antisense oligonucleotides, RNAi, shRNA, siRNA. In certain embodiments,the amount of detected free ubiquitin in the presence of a candidateagent is compared to a designated negative control, wherein a greateramount of free ubiquitin associated with the presence of the candidateagent indicates that the candidate agent decreases conjugation of theubiquitin protein or acts as a deubiquitinase, and a lesser amount offree ubiquitin associated with the presence of the agent indicates thatthe agent increases the conjugation of the ubiquitin protein ordecreases ubiquitination, e.g., inhibits a deubiquitinase. In someembodiments of the present invention, the screens are competition assaysthat utilize fluorescence intensity or anisotropy detection. In someembodiments, the screens utilize FRET detection. In some embodiments,measurements of free ubiquitin are made using a real-time assay, whereina change in the amount of free ubiquitin over time indicates thepresence of an agent that modulates ubiquitination. In some embodimentsof the invention, the amount of free ubiquitin-like protein isdetermined.

In certain embodiments, the screens are performed in a high throughputassay, e.g. wherein discrete wells of a multi-well plate include achimeric polypeptide sensor that preferentially binds to a freeubiquitin protein or a free ubiquitin-like protein and one or moreconjugated ubiquitin protein or conjugated ubiquitin-like protein, whichserves as a substrate of a candidate agent that modulates ubiquitinationor deubiquitination. Alternatively to, or in addition to, the conjugatedubiquitin protein or conjugated ubiquitin-like protein, the wells mayinclude a free ubiquitin protein and a ubiquitin protein substrate, or afree ubiquitin-like protein and a ubiquitin-like protein substrate,which may be conjugated by the free ubiquitin protein or the freeubiquitin-like protein. Different candidate agents are added todifferent wells, and their effect on the amount of free ubiquitinprotein or free ubiquitin-like protein. The assay may employ adetectably labeled chimeric polypeptide sensor that emits differentlywhen bound or unbound to ubiquitin or a ubiquitin-like protein, and/or adetectably labeled competitor ubiquitin protein or competitorubiquitin-like protein that emits differently when bound or unbound tothe sensor.

According to some embodiments of the invention, any chimeric polypeptidesensor of the invention can be incorporated into a screen as describedabove. This includes, but it not limited to chimeric polypeptidesensors: comprising two or more sequences that bind ubiquitin protein;comprising a detectable label; comprising two or more sequences thatbind ubiquitin-like protein; comprising a sequence that binds ubiquitinC-terminal; comprising a sequence that binds the ubiquitin hydrophobicpatch; comprising a sequence that binds to surface near ubiquitin Asp58;comprising a sequence that binds the ubiquitin C-terminal and a sequencethat binds the ubiquitin hydrophobic patch; comprising a sequence thatbinds to the ubiquitin C-terminal and a sequence that binds to thesurface near ubiquitin Asp58; comprising a sequence that binds to theubiquitin hydrophobic patch and a sequence that binds to the surfacenear ubiquitin Asp58; comprising a sequence that binds the ubiquitin Cterminal, a sequence that binds the ubiquitin hydrophobic patch, and asequence that binds to the surface near ubiquitin Asp58; comprising theZnF UBP binding domain from Isopeptidase T, comprising the Ruz domainfrom Rabex-5; comprising the ubiquitin interacting motif from Vps27;comprising the Ruz domain from Rabex-5 and the ubiquitin interactingmotif from Vps27; comprising the ZnF UBP domain from Isopeptidase T andthe Ruz domain from Rabex5; comprising the ubiquitin interacting motiffrom Vps27 and the Ruz domain from Rabex5; comprising the ZnF UBPbinding domain from Isopeptidase T, the Ruz domain from Rabex-5, and theubiquitin interacting motif from Vps27; comprising the ubiquitin bindingassociated domain from Dsk2; comprising the ZnF UBP binding domain fromIsopeptidase T and the ubiquitin associated domain from Dsk2; comprisingthe Ruz domain from Rabex-5 and the ubiquitin associated domain fromDsk2; comprising the ZnF UBP binding domain from Isopeptidase T, the Ruzdomain from Rabex-5, and the ubiquitin associated domain from Dsk2;comprising the ubiquitin interacting motif from S5a; comprising the ZnFUBP binding domain from Isopeptidase T, the Ruz domain from Rabex-5, theubiquitin interacting motif from S5a; further, this includes, but it notlimited to: tIVR, tIDR, tISR, tSR, and tIV. In some embodiments, theseinclude screens that comprise a ubiquitin protein or ubiquitin likeprotein tagged with a detectable label. In some embodiments, theseinclude screens that comprise a ubiquitin or ubiquitin-like proteintagged with a fluorophore. In some embodiments, these include screensthat comprise a chimeric polypeptide sensor with a detectable label anda ubiquitin protein or a ubiquitin like protein tagged with a detectablelabel. In some embodiments, these include screens comprising FRETimaging techniques.

In some embodiments, the present invention contains methods to determinethe level of ubiquitin proteins (e.g., total or free) that are presentin the serum of a patient. Stress or trauma can increase levels ofextracellular ubiquitin proteins present in serum. Furthermore, thereare extracellular ubiquitin receptors that can bind to the extracellularubiquitin receptors. An example of an extracellular ubiquitin receptoris CXCR4. CXCR4 is a G-protein coupled receptor that can bind toextracellular ubiquitin protein. Upon binding the extracellularubiquitin protein, CXCR4 becomes activated and can influence cellularprocesses. However, CXCR4 is only activated by ubiquitin proteins thatare intact. Extracellular ubiquitin proteins can be partially degradedin serum, and partially degraded ubiquitin proteins do not activateCXCR4. Of particular importance may be the presence of the C-terminaldomain on the ubiquitin proteins; an intact C-terminus of anextracellular ubiquitin is required to activate the CXCR4.

In some embodiments, the present invention includes methods to detectthe presence of free ubiquitin in serum. In some embodiments, the methodutilizes a chimeric polypeptide sensor comprising a polypeptide sequencethat binds to ubiquitin C-terminus. In some embodiments, the methodutilizes a chimeric polypeptide sensor wherein the chimeric polypeptidesensor has a selective binding affinity for free ubiquitin comprising anintact C-terminal as compared to partially degraded free ubiquitin orubiquitin-like proteins. In certain embodiments, the chimericpolypeptide sensor comprises a sequence that binds to the C-terminus ofa free ubiquitin protein. In some embodiments, the chimeric polypeptidesensor can be used in any of the assays of the invention describedherein. In some embodiments, these assays are competition assays. Insome embodiments, these assays comprise direct titration assays. In someembodiments, the chimeric polypeptide comprises a detectable label. Insome embodiments, the method utilizes a competitor ubiquitin proteintagged with a detectable label. In some embodiments, the detectablelabel is a fluorophore. In some embodiments, the method comprisesdetecting fluorescence intensity. In some embodiments, the methodcomprises detecting fluorescence anisotropy. In some methods, the methodcomprises FRET.

EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters that can be changed or modified to yieldessentially the same results.

Example 1 Design and Construction of Chimeric Polypeptides

This example demonstrates the design and subsequent generation ofchimeric polypeptides that bind to free ubiquitin proteins. Ubiquitinproteins can simultaneously interact with ubiquitin binding domains thatbind to non-overlapping regions of ubiquitin (see FIG. 1). Chimericpolypeptides were designed that comprise a domain that could bind to theubiquitin C terminus, a domain that binds the ubiquitin hydrophobicsurface patch, and a domain that binds to the surface of ubiquitin nearAsp58. These three binding domains were connected by two linker peptidesto generate chimeric polypeptides that act as sensors for free ubiquitinproteins (see FIG. 2). These three ubiquitin binding domains can beconnected by two linkers, resulting in a chimeric polypeptide thatsimultaneously binds the ubiquitin protein in three non-overlappingregions.

Using this strategy, two prototype chimeric polypeptide sensors wereconstructed. Both prototype chimeric polypeptides contain a domain thatbinds the ubiquitin C terminus, a domain that binds the ubiquitinhydrophobic patch, and a domain that binds to the surface of ubiquitinnear Asp58 that are connected by polypeptide linkers (see FIG. 3). Inthe first prototype (tIVR), the domain that binds to the C terminus ofubiquitin is the ZnF UBP domain of Isopeptidase T (IsoT^(ZnF)), thedomain that binds to the ubiquitin hydrophobic patch is theubiquitin-interaction motif from the Vps27 protein (Vps27^(UIM)), andthe domain that binds to the surface of ubiquitin near Asp58 is theRabex-5 ubiquitin binding zinc finger (Ruz). In the second prototype(tIDR), the IsoT^(ZnF) and Ruz domains are linked to the ubiquitinassociated (UBA) domain of the Dsk2 protein (Dsk2^(UBA)) which binds tothe ubiquitin hydrophobic patch. Amino acid sequences of the chimericpolypeptides are provided.

The amino acid sequences were as follows:

tIVR: (Underline shows each linker; SEQ ID NO: 1)MGSSHHHHHHSSGLVPRGSHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDPSLAEHLSHFGIDMLKMQKGSAAAEEAELDLKAAIQESLREAGGGSDLLCKKGCGYYGNPAWQGFCSKCWRE EYHKARQK tIDR:(Underline shows each linker; SEQ ID NO: 4)MGSSHHHHHHSSGLVPRGSHHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDPSLAEHLSHFGIDMLKMQKTGGSGGSGSGGSGPPEERYEHQLRQLNDMGFFDFDRNVAALRRSGGSVQGALDSLLNGGGGGSSGGGSDLLCKKGCGYYGNPAWQGFCSKCWREEYHKARQK

Structural models of the prototype chimeric polypeptide sensors bindingto the ubiquitin protein have been provided (see FIG. 4). The tIVRsensor is depicted with the IsoT^(ZnF), the Vps27^(UIM), and the Ruzdomains bound to the ubiquitin C terminus, the ubiquitin hydrophobicpatch, and the ubiquitin surface near Asp58, respectively. The tIDRsensor is depicted with the IsoT^(ZnF), the Dsk2UBA, and the Ruz domainsbound to the ubiquitin C terminus, the ubiquitin hydrophobic patch, andthe ubiquitin surface near Asp58, respectively.

The binding affinities of tIVR and tIDR to fluorophore-tagged ubiquitinwere determined. The values were calculated from assays incorporatingfluorophore-labeled ubiquitin. Experiments were performed withfluorescein, Atto532, and Alexa488-tagged ubiquitin in which maleimidederivatives of the fluorophores were attached to residue Cys20 (see FIG.5). The affinity of tIVR for free fluorophore-tagged ubiquitin proteinswas measured by detecting changes in fluorescence intensity orfluorescence anisotropy. These experiments took advantage of the factthat the fluorescent properties of fluorophore-tagged ubiquitin proteinchanges when it is bound to tIVR. Different concentrations of tIRV wereadded to fluorescein-tagged ubiquitin proteins. The affinity betweentIVR and Alexa488-tagged ubiquitin or Atto532-tagged ubiquitin wasmeasured by detecting change in fluorescence intensity. Differentconcentrations of tIVR were added to 50 μM Atto532-tagged ubiquitin or100 μM Alexa488-tagged tagged ubiquitin. Adding tIVR to thefluorophore-tagged ubiquitin increased the amounts of fluorophore-taggedubiquitin bound to tIVR, and thus decreased the fluorescence intensity(see FIG. 6, left). Affinity between fluorescein-tagged ubiquitin andtIDR was measured as the ratio of association and dissociation rates.Time-dependent changes in fluorescence anisotropy of 50 nMfluorescein-tagged ubiquitin were measured with and without 26 nM tIDRto measure association rates. Dissociation of 50 nM tIDR from itscomplex with 50 nM fluorescein-tagged ubiquitin was measured bydetecting change in fluorescence anisotropy upon addition of a 100-foldexcess of non-fluorescent ubiquitin (see FIG. 6, right).

The binding affinities of chimeric polypeptide sensors were compared tochimeric polypeptide sensors with different ubiquitin binding domains.The chimeric polypeptides were tagged with an Atto532 label. When theAtto532-tagged chimeric polypeptide is bound to a ubiquitin protein, thefluorescence intensity is altered. This allows for the fluorescentdetection of interactions between ubiquitin proteins and Atto532-taggedchimeric polypeptides. The affinities of Atto532-tagged chimericpolypeptides comprising three (tIVR L1 Cys), two (tIV L1 Cys), or one(IsoT^(ZnF) (S227C)) ubiquitin binding domains were determined (see FIG.7). Their amino acid sequences are as follows:

tIVR L1 Cys (SEQ ID NO: 2)MGSSHHHHHHSSGLVPRGSHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDPSLAEHLSHFGIDMLKMQKGSCAAAEEAELDLKAAIQESLREAGGGSDLLCKKGCGYYGNPAWQGFCSKCWR EEYHKARQK tIV L1 Cys(SEQ ID NO: 5) MGSSHHHHHHSSGLVPRGSHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVWSYDEDDMVLDPSLAEHLSHFGIDMLKMQKGSC AAAEEAELDLKAAIQESLREAIsoT^(ZnF) (S227C) (SEQ ID NO: 31)MPSSHHHHHHSSGLVPRGSHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGCGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDPSLAEHLSHFGIDMLKMQKThe bold “C” represents the fluorophore attachment site for eachchimeric polypeptide.

The determined K_(d) for each construct (see FIG. 7) demonstrate thestrong binding achieved by linking 3 non-overlapping ubiquitin bindingdomains.

This example shows that a chimeric peptide with strong affinity toubiquitin proteins has been constructed by linking more than one bindingdomain that can simultaneously bind to ubiquitin in non-overlappingregions.

Example 2 Binding Specificities of the Chimeric Polypeptides

The specificity of the chimeric polypeptide sensor for free ubiquitinwas demonstrated by comparing the binding affinities of the chimericpolypeptide sensors to free ubiquitin with the binding affinities toconjugated ubiquitin proteins or ubiquitin-like proteins (see FIG. 8). Acompetition assay was performed to determine the binding affinity oftIVR to free ubiquitin, conjugated ubiquitin, or nedd8, a ubiquitin-likeprotein. When bound to tIVR, the Atto532-tagged ubiquitin displayreduced fluorescent intensity when stimulated compared to Atto532-taggedubiquitin that is unbound to tIVR. The competitor proteins, freeubiquitin, conjugated ubiquitin (UB-GB1), or Nedd8 were added to a 10 nMtIVR and 10 nM Atto532-tagged ubiquitin mixture. Their amino acidsequences are as follows:

Ubiquitin (SEQ ID NO: 22)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG Nedd8 (SEQ ID NO: 23)MLIKVKTLTGKEIEIDIEPTDKVERIKERVEEKEGIPPQQQRLIVSGKQMNDEKTAADYKILGGSVLHLVLALRGG UB-GB1 (SEQ ID NO: 32)MGSSHHHHHHSSGLVPRGSHMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGQYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVT

Since Atto532-tagged ubiquitin displaced from tIVR has enhancedfluorescence emission, increased fluorescence intensity indicatesbinding of competitor proteins to tIVR. These data indicated that thetIVR polypeptide has an approximately 3000-fold higher affinity for freeubiquitin than for conjugated ubiquitin or Nedd8.

While tIVR shows a higher affinity to free ubiquitin than to conjugatedubiquitin, it has similar binding affinities for different kinds of freepolyubiquitin. A competition assay was performed testing 0.8 nMAtto532-tagged ubiquitin and 6 nM tIVR. Fluorescence intensity wasmeasured as different concentrations of different linkage types ofpolyubiquitin proteins were added. The binding affinities of eachlinkage-type polyubiquitin are listed in a table, and all K_(d) valuesfell within a range of 27-48 nM (see FIG. 9). This demonstrates thattIVR can provide a readout of free polyubiquitin without bias regardinglinkage type.

In addition to the binding domains, the linkers of the chimericpolypeptide can also influence the binding specificity of the chimericpolypeptide. Reducing the length of linker 1 (SEQ ID NO: 30) connectingthe domain that binds the ubiquitin C terminus with the domain thatbinds the ubiquitin hydrophobic patch increases the binding of tIVR tofree ubiquitin compared to a ubiquitin-protein conjugate (see FIG. 10).Further analysis showed that a minimal length for linker 1 helps thechimeric polypeptide sensors achieve more selectivity for free ubiquitinover conjugated ubiquitin (see FIG. 11). While tIVR with differentlength linker 1 regions could bind free ubiquitin with high affinity,tIVR with a short linker 1 (SEQ ID NO: 13) showed less affinity for aconjugated ubiquitin (Ub-GB1) than tIVR with a long linker 1 (SEQ ID NO:28; see FIG. 12).

Example 3 Use of Fluorescence Intensity as a Readout

The chimeric polypeptide sensors can be used with assays that measurefluorescence intensity to detect free ubiquitin. When chimericpolypeptide sensors bind a fluorophore-tagged ubiquitin protein, thefluorescence intensity is reduced compared to when thefluorophore-tagged ubiquitin is unbound. Alternatively, when afluorophore-tagged chimeric polypeptide sensor is bound to a ubiquitinprotein, the fluorescence intensity is increased compared to when thechimeric polypeptide is unbound (see FIG. 13).

Fluorophore-tagged ubiquitins have altered fluorescence intensity whenthey are bound to tIVR (see FIG. 14). The fluorescence intensity offluorescein, Alexa488, or Atto532-tagged ubiquitin in the presence oftIVR compared to the absence of tIVR was experimentally determined.Fluorescence intensities of fluorescein, Alexa488, or Atto532-taggedubiquitin in the presence or absence of tIVR is graphed; complexformation with tIVR results in decreased fluorescence. Conversely,fluorescence of Atto465, Atto532, and Alexa488-tagged tIVR exhibitedgreater fluorescence intensity when bound to ubiquitin.

Another chimeric polypeptide sensor, Atto532-tISR L1 Cys2, wasconstructed. This chimeric polypeptide comprises three binding domains.Atto532-tISR L1 Cys2 shows a large fluorescence intensity change uponbinding to free ubiquitin proteins. In this chimeric polypeptide sensor,L1 Cys2 is the linker between the IsoT^(ZnF) and the UIM and has a Cysresidue conjugated with the fluorophore. Relative fluorescence intensityof Atto532-tISR L1 Cys2 was measured following the addition of differentconcentrations of free ubiquitin proteins or conjugated ubiquitinproteins. Atto532-tISR L1 Cys2 showed an 8-fold increase in fluorescenceintensity when bound to free ubiquitin protein over a range of 0-1000 nMfree ubiquitin (see FIG. 15). There was little change in fluorescenceintensity across the same concentrations of conjugated ubiquitin. Theseexperiments demonstrate that Atto532-tISR L1 Cys2 has a largefluorescence change and a broad dynamic range for free ubiquitindetection.

The changes in fluorescence intensity measured during a competitionassay with fluorophore-tagged ubiquitin protein and tIVR were furtherexamined. Fluorescence intensity of emissions across 500 nm to 550 nmwavelengths of 3 nM fluorescein-tagged ubiquitin with 3 nM tIVR wasmeasured. Concentrations ranging from 0 to 3000 nM of untagged freeubiquitin were added to the mixture. Fluorescence intensity increasedwith higher concentrations of free ubiquitin. Further, there was noshift observed in the fluorescence emission wavelength due to additionsof the free ubiquitin (see FIG. 16). These results demonstrate thatfluorescence intensity is a reliable readout for assays involvingchimeric polypeptide sensors.

Example 4 Use of Anisotropy as a Readout

Anisotropy is another aspect of fluorescence signaling that can be usedwith chimeric polypeptide sensors as a signal to detect free ubiquitinproteins or ubiquitin-like proteins. As is the case with fluorescenceintensity, changes in anisotropy can be detected from the fluorophorewhen the chimeric polypeptide sensor and ubiquitin change between boundand unbound states.

Increased anisotropy can be detected from fluorophore-tagged ubiquitinprotein when it binds to chimeric polypeptide sensor (see FIG. 17).Concentrations of tIVR (0-10 nM) were added to mixtures with 150 pMfluorescein-tagged ubiquitin proteins and fluorescence anisotropy wasmeasured. Fluorescence was stimulated with polarized light at a 492 nmwavelength and polarized light emission was measured at 518 nm.Increasing the concentration of tIVR increased the anisotropy measuredin the mixtures as the fluorescein-tagged ubiquitin proteins were boundby tIVR.

Anisotropy can be measured to determine free ubiquitin in competitivebinding assays. Free ubiquitin protein was added to a mixture of 20 nMfluorescein-tagged ubiquitin proteins and 20 nM tIVR and anisotropy wasmeasured. Adding the free ubiquitin proteins to the mixture displacedfluorophore-tagged ubiquitin proteins from the complex with tIVR andreduced the anisotropy measured from the mixture (see FIG. 18). Thecurve generated from this experiment can be used as a calibrationstandard in assays to measure free ubiquitin proteins in experimentalsamples.

Example 5 Use of Fluorescence Resonance Energy Transfer (FRET) as aReadout

The chimeric polypeptide sensors can be used with fluorescence resonanceenergy transfer (FRET) to detect free ubiquitin proteins. A competitionassay was designed incorporating fluorescein-tagged ubiquitin proteinwith a modified tIVR tagged with a quencher. The amino acid sequence ofthe modified iIVR is as follows:

tIVR(R218C) (Bold indicates the attachment site of thequencher; SEQ ID NO: 3)MGSSHHHHHHSSGLVPRGSHMKQEVQAWDGEVRQVSKHAFSLKQLDNPARIPPSGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDPSLAEHLSHFGIDMLKMQKTGGSGGSGSAAAEEAELDLKAAIQESLREAGGGSSGGGSDLLCKKGCGYYGNPAWQG FCSKCWREEYHKACQK

The tIVR polypeptide can utilize fluorescence resonance energy transfer(FRET) to detect free ubiquitin proteins in a competition assay.Fluorescein-tagged ubiquitin proteins were combined in a mixture withthe tIVR conjugated the quencher. When the fluorescein-tagged ubiquitinproteins are bound to the tIVR (left), the fluorescence intensity of thefluorophore-tagged ubiquitin proteins is reduced by the quencherconjugated to the tIVR. Additionally, the tIVR itself also contributesto the reduction of the fluorescence. Adding free ubiquitin proteinsdisplaces bound fluorophore-tagged ubiquitin proteins, thus increasingthe fluorescence intensity of the mixture (see FIG. 19). This experimentdemonstrates that FRET is a feasible method to detect free ubiquitinproteins in conjunction with the chimeric polypeptide sensors. The curvegenerated from this experiment can be used as a calibration standard inassays to measure free ubiquitin proteins in complex biological samples.

Example 6 Deubiquitinase Assays with the Chimeric Polypeptides

The chimeric polypeptide sensors can be used to perform a deubiquitinaseassay in real-time. This was validated by an experiment monitoring thedeubiquitinase enzyme activity of YUH1 protein. The deubiquitinaseenzyme activity was measured in a real-time assay (see FIG. 20). Releaseof ubiquitin by YUH1, a deubiquitinase enzyme, was measured as afluorescence intensity change. Fluorescence intensity of a mixturecontaining YUH1, 500 nM of ubiquitin-D77, 10 nM fluorescein-taggedubiquitin, and 10 nM tIVR was measured over time. Activity of YUH1released ubiquitin proteins from conjugation to aspartic acid (i.e., theD77 residue), and thus increased the concentration of free ubiquitinprotein. The free ubiquitin proteins displaced bound fluorescein-taggedubiquitin, resulting in increased fluorescence intensity that wasdetected over time. This experiment demonstrates that real-timedeubiquitinase assays can be performed with the chimeric polypeptidesensors.

This was further demonstrated in a second experiment (see FIG. 21). Areal-time deubiquitinase assay using Atto532-tagged ubiquitin proteinsand tIVR was performed. A mimic of polyubiquitinated-protein substrateswas digested with 25 nM Usp2cc or 3 μM OTUB1, two deubiquitinaseenzymes, with or without UbcH5c in the presence of 1 nM Atto532-taggedubiquitin protein and 1 nM tIVR to detect free ubiquitin released by thedeubiquitinases. OTUB1 activity is upregulated by UbcH5c, and this wasdetected by the experiment (see FIG. 21). The amino acid sequences forthe polypeptides used in the experiment are as follows:

Ub5-Ovomucoid sequence is same as the one used in Yao et al. (2002)Nature vol. 419.

OTUB1 (SEQ ID NO: 29)MAAEEPQQQKQEPLGSDSEGVNCLAYDEAIMAQQDRIQQEIAVQNPLVSERLELSVLYKEYAEDDNIYQQKIKDLHKKYSYIRKTRPDGNCFYRAFGFSHLEALLDDSKELQRFKAVSAKSKEDLVSQGFTEFTIEDFHNTFMDLIEQVEKQTSVADLLASFNDQSTSDYLVVYLRLLTSGYLQRESKFFEHFIEGGRTVKEFCQQEVEPMCKESDHIHIIALAQALSVSIQVEYMDRGEGGTTNPHIFPEGSEP KVYLLYRPGHYDILYKUBCH5C (SEQ ID NO: 33)MALKRINKELSDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLVPEIARIYKTDRDKYNRISREWTQKYAMUsp2cc sequence is same as the one used in Catanzariti et al., ProteinSci. (2004) 13:1331

Example 7 Chimeric Polypeptides and Ubiquitin-Like Proteins

This example shows that chimeric polypeptides can preferentially bindfree ubiquitin-like proteins. A chimeric protein (tIV) containing twoubiquitin binding domains, IsoT^(ZnF) and Vps27^(UIM), binds to Nedd8with a K_(D) of 9.4 μM. Increasing concentrations of free Nedd8 proteinwere added to Atto532-tIV, and the fluorescence intensity was measured(see FIG. 22). Based on the binding interactions of Vps27UIM and Nedd8,tIV is predicted to have a greater binding affinity for free Nedd8protein than for conjugated Nedd8 protein. This experiment demonstratesthat chimeric polypeptides can be constructed to detect freeubiquitin-like protein.

Example 8 Design and Construction of Chimeric Polypeptides

This example demonstrates the design and subsequent generation ofchimeric polypeptides that bind to conjugated and free ubiquitinproteins. A single ubiquitin protein can simultaneously interact withmultiple ubiquitin binding domains that bind to non-overlapping regionsof ubiquitin (see FIG. 1). Chimeric polypeptides were designed thatcomprise a domain that binds the ubiquitin hydrophobic patch and adomain that binds to the surface of ubiquitin near Asp58 that areconnected by a linker. These binding domains were linked to generatechimeric polypeptides that act as sensors for conjugated or freeubiquitin proteins (see FIG. 23). These result in a chimeric polypeptidethat simultaneously binds ubiquitin proteins in two non-overlappingregions (see FIG. 24).

Using this strategy, a prototype chimeric polypeptide sensor wasconstructed (see FIG. 25). In the prototype (tSR), the domain that bindsto the ubiquitin hydrophobic patch is the ubiquitin-interaction motiffrom the S5A protein (S5a^(UIM)) and the domain that binds to thesurface of ubiquitin near Asp58 is the Rabex-5 ubiquitin binding zincfinger (Ruz).

The amino acid sequence is as follows:

tSR: (SEQ ID NO: 7) MPSSHHHHHHSSGLVPRGSHMTEEEQIAYAMQMSLREAGGGSDLLCKKGCGYYGNPAWQGFCSKCWREEAHKCAAERAAAE

Structural models of the prototype chimeric polypeptide sensor bindingto the ubiquitin protein have been provided. The tSR sensor is depictedwith the S5a^(UIM) and the Ruz domains bound to the ubiquitinhydrophobic patch and the ubiquitin surface near Asp58, respectively.

The binding affinities of tSR were determined (see FIG. 25). Acompetition assay was performed where free ubiquitin proteins, differentkinds of conjugated free ubiquitin proteins, and Nedd8 were added tomixtures of 5 nM Atto532-tagged ubiquitin protein and 600 nM tSR. Therelative fluorescence was measured. While tSR had similar bindingaffinities for unconjugated ubiquitin proteins and conjugated ubiquitinproteins, tSR had specificity against Nedd8. The Ki values forunconjugated ubiquitin proteins and the different kinds of conjugatedfree ubiquitin proteins are provided in a table (see FIG. 25). Theseresults indicate that the tSR chimeric peptide universal sensor does notdiscriminate among free ubiquitin or polyubiquitins conjugated atdifferent linkages.

The tSR can be used for direct titration experiments (see FIG. 26). Therelative fluorescence intensity changes of Alexa488-tagged tSR whenubiquitin is added at varying concentrations. The relative fluorescenceof 10 nM Alexa488 tSR was measured with different concentrations ofunconjugated ubiquitin protein, conjugated ubiquitin protein, or Nedd8.Binding of unconjugated ubiquitin protein and conjugated ubiquitinprotein, but not Nedd8 protein, to Alexa488-tagged tSR was detected.These results demonstrate that the tSR chimeric polypeptide showsselective binding to ubiquitin proteins over ubiquitin-like proteins.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin any Application Data Sheet, are incorporated herein by reference, intheir entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1.-201. (canceled)
 202. A method of determining an amount of a freeubiquitin protein in a sample, the method comprising: (a) contactingsaid sample for a period of time with a chimeric polypeptide thatpreferentially binds to said free ubiquitin protein as compared to aconjugated ubiquitin protein, wherein said chimeric polypeptidecomprises: (i) a first polypeptide that binds a ubiquitin proteinmonomer and a second polypeptide that binds said ubiquitin proteinmonomer, wherein said first and second polypeptides bind non-overlappingregions of said ubiquitin protein monomer, wherein said firstpolypeptide binds to the C-terminus of said ubiquitin protein monomerand comprises a zinc finder binding domain of Isopeptidase T; whereinsaid second polypeptide binds to the ubiquitin hydrophobic patch of saidubiquitin protein monomer and comprises a ubiquitin binding domainselected from the group consisting of: Ubiquitin Associated domain(UBA), a Ubiquitin Interacting Motif (UIM), a double-sidedubiquitin-interacting motif (DUIM), a Motif Interacting with Ubiquitin(MIU), a coupling of ubiquitin conjugation to ER degradation (CUE), aGolgi-localized, Gamma-ear-containing, Arf-binding (GAT), a Jun kinaseactivation domain binding/Mpr1p and Pad1p N-termini (Jab1/MPN), a Np14 azinc finger (NZF), a ubiquitin-binding zinc finger (UBZ), a Ubiquitinbinding surface (UBS), and a Ubiquitin binding motif (UBM); (ii) a firstlinker, wherein said first linker connects said first and secondpolypeptides; (b) determining: (i) an amount of said free ubiquitinprotein bound to said chimeric polypeptide; or (ii) an amount of a freecompetitor ubiquitin protein bound to said chimeric polypeptide, whereinif step (b) (ii) is performed, the method further comprises step (c);(c) contacting said sample with said free competitor ubiquitin proteinprior to step (b); wherein (i) said chimeric polypeptide, (ii) said freecompetitor ubiquitin protein, or (iii) each of said chimeric polypeptideand said free competitor ubiquitin protein further comprises adetectable label; thereby determining the amount of said free ubiquitinprotein in said sample.
 203. The method of claim 202, wherein said firstpolypeptide comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO: 31, amino acidresidues 1-147 of the amino acid sequence of SEQ ID NO: 1, and aminoacid residues 1-148 of the amino acid sequence of SEQ ID NO:
 2. 204. Themethod of claim 202, wherein said second polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,and SEQ ID NO:
 42. 205. The method of claim 202, wherein (i) said firstpolypeptide comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO: 31, amino acidresidues 1-147 of the amino acid sequence of SEQ ID NO: 1, and aminoacid residues 1-148 of the amino acid sequence of SEQ ID NO: 2; and (ii)said second polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:
 42. 206. Themethod of claim 205, wherein said chimeric polypeptide furthercomprises: (c) a third polypeptide that binds to said ubiquitin proteinmonomer, wherein said first polypeptide, said second polypeptide, andsaid third polypeptide bind to non-overlapping regions of said ubiquitinprotein monomer, and; (d) a second linker, wherein said second linkerconnects said third polypeptide and said second polypeptide, whereinsaid third polypeptide binds to the surface of said ubiquitin proteinmonomer near Asp58 and comprises the Ruz domain of Rabex-5.
 207. Themethod of claim 205, wherein said chimeric polypeptide furthercomprises: (c) a third polypeptide that binds to said ubiquitin proteinmonomer, wherein said first polypeptide, said second polypeptide, andsaid third polypeptide bind to non-overlapping regions of said ubiquitinprotein monomer, and; (d) a second linker, wherein said second linkerconnects said third polypeptide and said first polypeptide, wherein saidthird polypeptide binds to the surface of said ubiquitin protein monomernear Asp58 and comprises the Ruz domain of Rabex-5.
 208. The method ofclaim 206, wherein said third polypeptide comprises the amino acidsequence of SEQ ID NO:
 10. 209. The method of claim 207, wherein saidthird polypeptide comprises the amino acid sequence of SEQ ID NO: 10.210. The method of claim 202, wherein said detectable label is afluorophore and the detecting comprises measuring fluorescence intensityor fluorescence anisotropy.
 211. The method of claim 202, furthercomprising: (d) comparing the amount of the bound free ubiquitin proteinto a predetermined value or to a control value.
 212. The method of claim202, wherein step (b) comprises determining the amount of the freeubiquitin competitor protein bound to said chimeric polypeptide at twoor more time points during said period of time.
 213. The method of claim212, further comprising: (d) comparing the amount of said bound freeubiquitin protein to a predetermined value or to a control value at twoor more time points during the period of time.
 214. The method of claim202, comprising: (a) contacting said sample for said period of time withsaid chimeric polypeptide that preferentially binds to said freeubiquitin protein as compared said conjugated ubiquitin protein; (b)contacting said sample with said free competitor ubiquitin protein; and(c) determining the amount of said free competitor ubiquitin proteinbound to said chimeric polypeptide.
 215. The method of claim 214,further comprising: (d) comparing the amount of the free competitorubiquitin protein bound to said chimeric polypeptide to a predeterminedvalue or to a control value.
 216. The method of claim 214, wherein step(c) comprises determining the amount of said ubiquitin competitorprotein bound to said chimeric polypeptide at two or more time pointsduring said period of time.
 217. The method of claim 216, furthercomprising: (d) comparing the amount of said ubiquitin competitorprotein to said chimeric polypeptide to a predetermined value or acontrol value at two or more time points during said period of time.218. The method of claim 202, wherein said sample is a biological samplederived from (a) biological tissue that optionally comprises eukaryoticcells or (b) cultured cells that optionally comprises eukaryotic cells.219. The method of claim 202, wherein (i) said chimeric polypeptidecomprises said detectable label; or (ii) said free competitor ubiquitinprotein comprises said detectable label.
 220. The method of claim 214,wherein said free ubiquitin competitor protein is tagged with a firstdetectable label, and said chimeric polypeptide is tagged with a seconddetectable label.
 221. The method of claim 220, wherein said firstdetectable label and said second detectable label are suitable forperforming fluorescence resonance energy transfer (FRET), and wherein(a) said first detectable label is a quencher and said second detectablelabel is a fluorophore; or (b) said first detectable label is afluorophore and said second detectable label is a quencher.